Methods of increasing bone mass in a subject comprising inhibiting DKK1

ABSTRACT

The present invention is drawn to understanding lytic bone diseases. In this regard, the present invention discloses mechanism by which Wnt signaling antagonist inhibits bone differentiation. Also disclosed herein are methods to control bone loss, treat bone disease and prevent tumor growth in bones of individual.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation under 35 U.S.C. 120 U.S. Ser. No.12/008,771, filed Jan. 14, 2008, now U.S. Pat. No. 8,124,087, which is acontinuation-in-part under 35 U.S.C. 120 of U.S. Ser. No. 11/588,008,filed Oct. 26, 2006, now U.S. Pat. No. 7,811,750, which is acontinuation-in-part under 35 U.S.C. 120 of U.S. Ser. No. 11/176,739,filed Jul. 7, 2005, now U.S. Pat. No. 7,642,238, which is acontinuation-in-part under 35 U.S.C. 120 of U.S. Ser. No. 10/727,461,filed Dec. 4, 2003, now U.S. Pat. No. 7,459,437, which claims benefit ofpriority under 35 U.S.C. 119(e) of provisional application U.S. Ser. No.60/431,040, filed Dec. 5, 2002, the entirety of all of which are herebyincorporated by reference.

FEDERAL FUNDING LEGEND

This invention was made with governmental support under Grant NumbersCA93897, CA55819 and CA97513 awarded by the National Cancer Institute.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the study of multiplemyeloma. More specifically, the present invention relates to theidentification and validation of molecular determinants of myeloma bonedisease through comparative global gene expression profiling andemployment of the SCID-rab mouse model for primary myeloma. Further,this invention relates to methods of treatment of bone disease bystimulating bone formation and reducing bone loss via targetingmolecular determinants identified by the global gene expressionprofiling.

2. Description of the Related Art

Multiple myeloma (MM) is a rare, yet incurable malignancy of terminallydifferentiated plasma cells (PC) that affects approximately 15,000persons per year in the United States, and represents the second mostcommon hematopoietic malignancy. Multiple myeloma represents 13% of alllymphoid malignancies in the white population and 31% of lymphoidmalignancies in the black population. The malignant plasma cells home toand expand in the bone marrow causing anemia and immunosuppression dueto loss of normal hematopoiesis.

Multiple myeloma is also associated with systemic osteoporosis and localbone destruction leading to debilitating bone pain and susceptibility tofractures, spinal cord compression and hypercalcemia. Myeloma is theonly hematological malignancy consistently associated with lytic bonedisease and local bone destruction is limited to areas adjacent toplasma cells, suggesting that the malignant plasma cells secrete factorsthat enhance osteoclast function and/or osteoblast anergy. Theprevalence of bone disease varies with the presentation of myeloma, fromsmoldering myeloma, often without bone involvement, to solitaryplasmacytoma, to diffused or focal multiple myeloma where systemiclosses of bone mineral density or focal lytic bone lesions are seen inapproximately 80% of patients.

In recent years, it has become evident that lytic bone disease is notonly a consequence of myeloma, but that it is intricately involved inpromoting disease progression. Change in bone turnover rates predictsclinical progression from monoclonal gammopathy of undeterminedsignificance (MGUS) to overt myeloma by up to 3 years. While initiallyosteoclast and osteoblast activity are coupled, the coupling is lostwith disease progression. Osteoclast activity remains increased andosteoblast activity is diminished, with lytic bone disease as theconsequence. Studies in the 5T2 murine myeloma and the SCID-hu model forprimary human myeloma demonstrated that inhibition of osteoclastactivity is associated with inhibition of myeloma growth and reductionof myeloma tumor burden. These studies support reports that inhibitionof bone resorption with bisphosphonates had an anti-myeloma effect.

Whereas the biology of osteoclasts in myeloma-associated lytic bonedisease has been investigated intensively, little is known about thedisease-associated changes in osteoblast activity and their underlyingmechanisms. It has been suggested that in myeloma, the ability ofmesenchymal stem cells to differentiate into the osteogenic lineage isimpaired. However, the mechanisms responsible for such impairment havenot been elucidated.

The Wnt signaling pathway is involved in both normal skeletogenesis andcancer related bone disease. The first link between Wnt signaling andhuman bone disease came from observations that inactivating mutations inthe Wnt co-receptor, LRP5, causes the osteoporosis-pseudoglioma syndrome(OPPG) (Gong et al., 2001). The canonical Wnt signaling pathway isregulated by large number of antagonists, including the DKK family andsecreted frizzled-related protein (SRFPs). To date, four Dkk proteinshave been identified in mammals (Kawano and Kyota, 2003), among whichDkk1 and Dkk2 have been well characterized. Subsequently it was shownthat mutations in LRP5 that causes a high bone mass phenotype weredistinct from those seen in osteoporosis-pseudoglioma syndrome andprevented binding of Dickkopf-1 (DKK1), a soluble inhibitor of Wnt andhigh affinity ligand for LRP5 (Boyden et al., 2002; Little et al.,2002). DKK1, antagonizing the canonical Wnt pathway by binding to LRP5/6and Kremen (Bafico et al., 2001; Mao et al., 2002; Mao et al., 2001),blocks maturation of osteoblasts and formation of mineralized matrix(Baron and Rawadi, 2007; van der Horst et al., 2005).

Additionally, over-expression of DKK1 in transgenic mice leads todecreased bone mass (Baron and Rawadi, 2007), while deletion of a singleallele of DKK1 in mouse osteoblasts results in increased bone formationand bone mass (Morvan et al., 2006). The osteolytic prostate cancer linePC-3, when transfected with shRNA targeting DKK1, reverted to anosteoblastic phenotype. In addition, transfection of DKK1 into theosteoblastic prostate cancer cell line C4-2B, which normally induces amix of osteoblastic and osteolytic lesions, caused the cells to developosteolytic tumors in SCID mice. Thus, the role of DKK1 in promoting bonelesion development appears not to be limited to MM, but has also beenindicated in prostate cancer.

In addition to inhibiting osteoblastogenesis, elevated DKK1 levels mayenhance osteoclastogenesis. Thus, bone destruction, a cardinal featureof multiple myeloma (MM) may result from uncoupling of osteoclast andosteoblast activities (Bataille et al., 1991; Roodman, 2004; Taube etal., 1992). Osteoclasts are activated by binding of receptor activatorof nuclear factor kappa B ligand (RANKL) (Anderson et al., 1997; Kong etal., 1999; Lacey et al., 1998) to its cognate receptor, RANK, whileosteoprotegerin (OPG) (Simonet et al., 1997) (a soluble member of thetumor necrosis receptor super-family) acts as a naturally occurringdecoy receptor that competes with RANK for binding of RANKL (Suda etal., 1999). MM cells likely stimulate expression of RANKL and suppressexpression of OPG by osteoblasts or their progenitors (Giuliani et al.,2001; Pearse et al., 2001). Increased serum levels of RANKL anddecreased levels of OPG have been associated with a poor prognosis in MM(Terpos et al., 2003). Restoring the RANKL/OPG imbalance by RANKLantagonist or recombinant OPG not only reduce MM-associated bone lesionsbut also halt disease progression in animal models (Pearse et al., 2001;Vanderkerken et al., 2003; Yaccoby et al., 2002; Oyajobi et al., 2001).

Mechanistically, regulation of osteoclastogenesis by osteoblast-derivedOPG (Glass et al., 2005; Holmen et al., 2005; Jackson et al., 2005) andRANKL (Holmen et al., 2005; Galli et al., 2006; Spencer et al., 2006)involves Wnt signaling, a pathway that is regulated by a large number ofantagonists, including members of the Dickkopf family (Morvan et al.,2006), the family of secreted frizzled-related protein (sRFPs) (Finch etal., 1997; Kawano and Kypta., 2003), and sclerostin (Semenov et al.,2005). Osteolytic bone lession (OBL) in MM cells could be linked to DKK1secretion by tumor cells (Tian et al., 2003; Giuliani et al., 2007;Haaber et al., 2007; Politou et al., 2006), inhibiting canonical Wnt inand differentiation of osteoblasts. Blocking DKK1 with a neutralizingantibody prevented MM-induced bone resorption in the SCID-rab model(Yaccoby et al., 2007). Although each appear to play an role in OBL,whether DKK1 might influence RANKL/OPG expression in myeloma has neverbeen established.

The prior art is deficient in methods to diagnose and treat multiplemyeloma bone diseases. Furthermore, the prior art is also deficient inunderstanding the disease-associated changes in osteoblast activity andthe underlying mechanisms in multiple myeloma associated lytic bonediseases. The present invention fulfills this longstanding need anddesire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a method of controlling bone lossin an individual. This method comprises the step of inhibiting a Wntsignaling antagonist at the nucleic acid or protein level. The presentinvention is also directed to a method of treating bone disease in anindividual, comprising the step of administering to the individual apharmacologically effective amount of an inhibitor of a Wnt signalingantagonist. Such a step results in blocking of induction of Wnt ligand,restoring the RANKL/OPG levels or both.

The present invention is also directed to a method of inhibiting tumorgrowth in bone of an individual. Such a method comprises the step ofblocking the activity of DKK1.

The present invention is also directed to a method of screening for acompound that controls bone loss and inhibits human myeloma growth. Sucha method comprises engrafting human myeloma cells in a rabbit boneimplanted in a SCID-rab mouse. This is followed by administration of acandidate compound to the mouse. Subsequently, bone mineral density ofthe implanted bone and level of serum human monoclonal immunoglobulin inthe mouse is compared with a control mouse that has not received thecompound. An increase in the bone mineral density and a decrease in thelevel of the serum immunoglobulin in the treated mouse compared to thecontrol mouse indicates that the compound controls bone loss andinhibits human myeloma growth. The present invention is further directedto a method of inhibiting multiple myeloma growth. Such a methodcomprises blocking of the DKK1 activity.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention as well as others which will become clear areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate preferred embodiments of the invention and therefore are notto be considered limiting in their scope.

FIGS. 1A-1B show global gene expression patterns reflecting bone lesionsin myeloma. FIG. 1A shows clusterview of normalized expression levels of57 genes identified by logistic regression analysis as beingsignificantly differentially expressed in malignant plasma cells frompatients with no (n=36) and 1+MRI focal lesions (n=137) (P<0.0001). The28 genes exhibiting elevated expression in plasma cells from patientswith 1+MRI lesions are ordered from top to bottom based on rank ofsignificance. Likewise the 30 genes showing significant elevation inpatients with no MRI-lesions are ordered from bottom to top based onsignificance rank. Gene symbols (Affymetrix probe set identifiers whenthe gene is unnamed) are listed to the left. Normalized expressionscales range from −30 (blue) to +30 (red) as indicated below the datadisplay. The four genes remaining significant after permutationadjustment are underlined. FIG. 1B shows a bar graph of DKK1 geneexpression in plasma cells from normal bone marrow (BPC), patients withmonoclonal gammopathy of undetermined significance (MGUS), Waldenström'smacroglobulinemia (WM), and multiple myeloma (MM) presented on thex-axis. MM samples are broken down into three bone lesion groups: noMRI/no x-ray lesions, 1+MRI/no x-ray lesions, and 1+MRI/1+x-ray lesions.The Affymetrix Signal, a quantitative measure of gene expression derivedfrom MAS 5.01, is indicated on the y-axis. DKK1 gene expression level ineach sample is indicated by a bar, with the height of the barproportional to gene expression intensity. Samples are ordered from thelowest to highest DKK1 gene expression from left to right on the x-axis.The number of samples in each group is indicated below each groupdesignator. Statistics for comparisons between the MM subgroups areindicated in the text.

FIG. 2 shows RHAMM was up-regulated in multiple myeloma patients withbone lesions.

FIG. 3 shows RHAMM rarely present in normal plasma cells and monoclonalgammopathy of undetermined significance (MGUS), but it was present invirtually all human myeloma cell lines.

FIG. 4 shows securin was up-regulated in multiple myeloma patients withbone disease.

FIG. 5 shows MIP-1a and CCR1 were “spike” genes in multiple myeloma, butthey were not correlated with lytic lesions. Black bar: CCR1; gray bar:MIP-1a.

FIG. 6 shows MIP-1a was expressed at low level in normal plasma cells(PC).

FIG. 7 shows the expression of WNT antagonist DKK-1 in multiple myelomawith bone lesions.

FIG. 8 shows the expression of WNT decoy receptor FRZB in multiplemyeloma with lytic bone lesions.

FIG. 9 shows the expression of DKK-1 and FRZB in multiple myeloma withlytic bone lesions. Black bar: DKK-1; gray bar: FRZB.

FIG. 10 shows FRZB was expressed in tonsil plasma cells. PBC, TBC,tonsil B cells; TPC, tonsil plasma cells; BPC, bone marrow plasma cells;WPC, WBC, CLL.

FIG. 11 shows DKK-1 was not expressed in normal B cells or plasma cells.PBC, TBC, tonsil B cells; TPC, tonsil plasma cells; BPC, bone marrowplasma cells; WPC, WBC, CLL.

FIG. 12 shows DKK-1 expression in monoclonal gammopathy of undeterminedsignificance (MGUS) was low relative to smoldering multiple myeloma(SMM) and newly diagnosed multiple myeloma (MM).

FIG. 13 shows FRZB was elevated in monoclonal gammopathy of undeterminedsignificance (MGUS), and had higher expression in smoldering multiplemyeloma (SMM) and newly diagnosed multiple myeloma (MM).

FIG. 14 shows the expression of DKK-1 and FRZB in monoclonal gammopathyof undetermined significance (MGUS) and smoldering multiple myeloma(SMM).

FIG. 15 shows low expression of DKK-1 in extramedullary disease.

FIG. 16 shows the expression of DKK-1 and FRZB tend to be higher inplasma cells from medullary PCT than those from iliac crest. PCT, FNA.

FIG. 17 shows the expression of DKK-1 and FRZB in fine needle aspiratesof medullary PCT.

FIG. 18 shows high expression of DKK-1 and FRZB in medullaryplasmacytoma.

FIG. 19 shows higher expression of DKK-1 in multiple myeloma withosteopenia.

FIG. 20 shows DKK-1 was not expressed in plasma cells from Waldenstrom'smacroglobulinemia.

FIG. 21 shows WNT5A was elevated in newly diagnosed multiple myeloma.

FIG. 22 shows WNT5A tends to be higher in multiple myeloma with lyticlesions.

FIG. 23 shows WNT5A was also elevated in monoclonal gammopathy ofundetermined significance (MGUS) and smoldering multiple myeloma (SMM).

FIG. 24 shows WNT10B tends to be lower in multiple myeloma with lyticlesions.

FIG. 25 shows WNT5A and WNT10B tend to be inversely correlated. Blackbar: WNT10B; gray bar: WNT5A.

FIG. 26 shows DKK-1 was present in an SK-LMS cell line.

FIG. 27 shows primary multiple myeloma synthesized DKK-1 protein.

FIG. 28 shows low DKK-1 expression in relapsed and primary refractorymultiple myeloma.

FIG. 29 shows endothelin receptor B was a “spike” gene in one third ofnewly diagnosed multiple myeloma.

FIG. 30 shows the expression of endothelin receptor B in monoclonalgammopathy of undetermined significance (MGUS) and smoldering multiplemyeloma. Normal plasma cells do not express endothelin receptor B.

FIG. 31 shows the involvement of endothelin receptor B in boneformation.

FIG. 32 shows DKK-1 expression after treatment with PS-341.

FIG. 33 shows DKK-1 expression after treatment with thalidomide in newlydiagnosed multiple myeloma.

FIG. 34 shows DKK-1 expression after treatment with IMiD.

FIG. 35 shows DKK-1 expression after treatment with dexamethsone innewly diagnosed multiple myeloma.

FIG. 36 shows downregulation of JUN and FOS in multiple myeloma cellsafter co-culture with osteoclasts.

FIG. 37 shows JUN & DKK-1 downregulation in osteoclast co-culture.

FIG. 38 shows WNT signaling in multiple myeloma bone disease.

FIG. 39 shows overexpression of DKK1 in low grade myeloma with the lossof expression with disease progression. Expression of DKK1 was examinedby immunohistochemistry of myeloma bone marrow biopsies. Serial sections(550× magnification) of bone marrow biopsies from myeloma patients withhigh (a-b) and low (c-d) DKK1 gene expression are presented. Slides arestained with H&E (a and c) or anti-DKK1 and secondary antibody (b andd). Use of secondary alone failed to stained cells (data not shown).Magnified images (1,200× magnification) are located in the upper leftcorner of each H&E image. Image a shows a myeloma with an interstitialpattern of involvement with plasma cells exhibiting low grade morphologywith abundant cytoplasm and no apparent nucleoli. Image b revealspositive staining of plasma cells in a interstitial pattern withanti-DKK1 antibody that was greatest adjacent to bone. Image c shows amyeloma with nodular or alliterative pattern with plasma cellsexhibiting high grade morphology with enlarged nuclei and prominentnucleoli. Image d reveals no positive staining of plasma with anti-DKK1antibody.

FIGS. 40A-40B show DKK1 protein in the bone marrow plasma is highlycorrelated with DKK1 gene expression and the presence of bone lesions.FIG. 40A shows the expression of DKK1 mRNA was detected by microarrayand DKK1 protein by ELISA in a total of 107 cases of newly diagnosedmyeloma. Results of both assays were transformed by the log base 2 andnormalized to give a mean of 0 and variance of 1. Each bar indicates therelative relationship of gene expression and protein expression in eachsample. There was a significant correlation between DKK1 mRNA in myelomaplasma cells and protein in bone marrow plasma (r=0.65, P<0.001). FIG.40B shows bar view of DKK1 protein levels in bone marrow plasma cellsfrom normal donors (BPC), patients with monoclonal gammopathy ofundetermined significance (MGUS), Waldenström's macroglobulinemia (WM),and multiple myeloma (MM) are presented on the x-axis. MM samples arebroken down into three bone lesion groups: no MRI/no x-ray lesions,1+MRI/no x-ray lesions, and 1+MRI/1+x-ray lesions. The DKK1 proteinconcentration (ng/ml) is indicated on the y-axis. To enable comparisonsof DKK1 protein levels in the lower ranges, 200 ng/ml was made themaximum value. This resulted in the truncation of a single sample withDKK1 concentration of 476 ng/ml. DKK1 protein level in each sample isindicated by a bar, with the height of the bar proportional to DKK1protein levels. Samples are ordered from the lowest to highest DKK1protein levels from left to right on the x-axis. The number of samplesin each group is indicated below each group.

FIGS. 41A-41B show recombinant DKK1 and MM plasma can block alkalinephosphatase production in BMP-2 treated C2C12 cells in a DKK1-dependentmanner. FIG. 41A shows alkaline phosphatase levels, a marker ofosteoblast differentiation (y-axis) were measured in C2C12 cells after 5days of culture in the presence of 5 percent fetal calf serum alone orwith BMP2, BMP2+DKK1, BMP2+DKK1+anti-DKK1, or BMP-2+DKK1+polyclonal IgG.Each bar represents the mean (±SEM) of triplicate experiments. Note thatactivity of alkaline phosphatase increased in the presence of BMP-2 andsignificant reduction of this protein by co-incubation with recombinantDKK1. Also note that anti-DKK1 antibody, but not polyclonal IgG canblock the repressive activity of DKK1. FIG. 41B shows alkalinephosphatase levels (y-axis) were tested in C2C12 cells after culturingthese cells for 5 days in 5 percent fetal calf serum alone or 50 ng/mlBMP-2±10 percent normal bone marrow plasma (NS) or BMP-2±10 percentmyeloma bone marrow plasma from 10 patients with newly diagnosed myeloma(sample identified provided), or BMP2±10 percent myeloma patientplasma+anti-DKK1 or goat polyclonal IgG. Each bar represents the mean(±SEM) of triplicate experiments. DKK1 concentration from each bonemarrow plasma samples was determined by ELISA and final concentrationsin culture after 1:10 dilution are indicated on the x-axis. Note thatsamples with >12 ng/ml DKK1 had an effect on alkaline phosphataseproduction. A star indicates P<0.05 in comparison to alkalinephosphatase in BMP2±10 percent normal human bone marrow plasma.

FIGS. 42A-42DD show that DKK1 blocks the osteoblast differentiation byblocking an endogenous Wnt signal being made by the osteoblastprecursor.

FIGS. 42A-42D show that Frizzled (Fz) and LRP5/6 mRNAs are expressed inosteoblast cells. Total RNA was extracted from indicated cell lines.RT-PCR was performed with mouse (FIG. 42A) and human (FIG. 42B) primersspecific for Fz1 through 10 and LRP5/6 (mouse, FIG. 42C); human, (FIG.42D), respectively. GAPDH was included as control.

FIGS. 42E-42I show that canonical Wnt signaling is functional inosteoblast (OB) cells. OB cells were treated with Wnt3a CM or control CMfor indicated time and cell lysate harvested. Lysate protein wassubjected to GST-E-cadherin assay and Immunoblotting analysis usinganti-_-catenin antibody (FIG. 42E) and anti-non-phosphorylated form(FIG. 42F). RT-PCR was performed using specific primers for indicatedmouse (FIG. 42G) and human (FIG. 42H) genes in indicated cell lines.C2C12 cells transfected using wild type (TOPflash) or mutant (FOPflash)LEF/TCF reporter luciferase constructs and pSV-b-galactosidase vector(transfection efficiency internal control) (FIG. 42I) were treated withWnt3a CM or control CM prior to determination of luciferase activity.Results are shown as mean±SD (n=3) and are representative of threeindependent experiments. ** P<0.01 versus control.

FIGS. 42J-42M show that Dkk-1 and MM patient sera inhibit Wnt3a inducedaccumulation of b-catenin in OB cells. In FIG. 42J, the indicated cellswere treated with recombinant Dkk-1 at indicated concentrations and thenstimulated with Wnt3a or control CM. proteins in cell lysate weresubjected to GST-E-cadherin assay and Immunoblotting analysis usinganti-non-phosphorylated b-catenin. In FIG. 42K, Dkk1 protein in celllysate (upper panel) and CM (middle panel) from OPM-2 clones expressingPEF6/V5-His-TOPO-Dkk-1 (Dkk1) or vector (EV) was confirmed byImmunoblotting blotting with anti-V5 antibody. In FIG. 42L, C2C12 cellswere incubated with Dkk1 or EV CM from OPM-2 transfected cells thentreated with Wnt3a or control CM. proteins in lysates were analyzed asin FIG. 42B using indicated antibody. In FIG. 42M, C2C12 cells wereincubated with sera from MM patients containing low (L1) or high (H1-4)concentrations of Dkk1 protein. Cells were then treated with Wnt3a orcontrol CM and lysates prepared and analyzed as in FIG. 42L.

FIGS. 42N-42P show that Dkk1 inhibits BMP-2 induced alkaline phosphataseactivity. C2C12 (FIG. 42N), Saos-2 (FIG. 42O), and hFOB1.19 (FIG. 42P)cells were cultured for 72 hrs in DMEM with 2% horse serum containingWnt3a, BMP-2, or Dkk1 either alone or in the indicated combinations.Cells were lysed and ALP activity measured and normalized to proteinconcentration. Data represent the mean±SD (n=3). *p<0.05, ** P<0.01,***P<0.001. *****<0.00001 versus control or BMP-2 versus BMP-2 plusDKK1.

FIGS. 42Q-42V show that blocking the canonical b-catenin pathwayinhibits TCF/LEF mediated transcription and BMP-2 induced alkalinephosphatase activity. In FIG. 42Q, C2C12 cells were transfected withdominant negative b-catenin (DN-b-Cat) or control (pcDNA4his) constructsand the expressions were confirmed by the indicated antibody byImmunoblotting analysis. In FIG. 42R, the positive clones wereco-transfected with wild type (TOPflash) or mutant (FOPflash) LEF/TCFreporter luciferase constructs. After transfection, the cells weretreated and subjected to luciferase assay as in FIG. 2 E. C2C12 pcDNAhisor DN-b-Cat clone #4 (FIG. 42S) and #5 (FIG. 42T) cells were cultured inDMEM containing 2% horse serum or 100 ng/ml of BMP-2 for 72 hrs afterwhich ALP activity was measured. In FIG. 42U, C2C12 cells weretransfected with empty vector or Dkk1- and Dkk2-expressing vectors andthe expression of the proteins were determined as in FIG. 42K. Thepositive clones and control vector were treated with BMP-2 and subjectedto ALP activity assay as in FIG. 42T. Results are shown as mean±SD (n=3)and are representative of three independent experiments (FIG. 42V). **P<0.01 and *** p<0.001 versus control.

FIGS. 42W-42Z show that silencing LRP5/6 mRNA blocks BMP-2 inducedalkaline phosphatase activity. C2C12 cells were transiently transfectedwith 1.0 mg/ml (FIG. 42W) or serial concentration of siRNA specific forLRP5 (FIG. 42X) or LRP6 (FIG. 42Y). Forty eight hrs after transfectionRNA was isolated and subjected to RT-PCR (FIG. 42W) or qPCR (FIGS. 42Xand 42Y). In FIG. 42Z, the cells transfected with 0.25 or 0.5 mg/ml ofsiRNA specific for LRP5 or LRP6 were cultured in medium containing 100ng/ml of BMP-2 in DMEM. Cells were lysed after 72 hrs and alkalinephosphatase activity determined. Data represent the mean±SD (n=3) ofrepresentative experiments. *p<0.05, **p<0.01, ***p<0.001 versuscontrol.

FIGS. 42AA-42BB show that Wnt3a mediated increase in b-catenin isindependent of BMP-2. In FIG. 42AA, C2C12, hFOB1.19, MG63, and Saos-2cells were treated with Wnt3a CM, control CM or 100 ng/ml of BMP2.Lysate protein was subjected to GST-E-cadherin assay and Immunoblottinganalysis by anti-b-catenin antibody as described in Materials andMethods. In FIG. 42BB, 50 mg aliquots of protein from cell lysates wereresolved on 8% SDS-PAGE and analyzed with the indicated antibodies.

FIG. 42CC shows C2C12 cells that were transfected with wild type(TOPflash) LEF/TCF reporter luciferase constructs. After transfection,the cells were treated with 100 ng/ml of Wnt3a, BMP2 (100 ng/ml) orcombined with Wnt3a and BMP2 or with Dkk1 (100 ng/ml) for 24 hours andthen subjected to luciferase assay. Results are shown as mean±SD (n=3)and representative of three independent experiments. **P<0.01 versuscontrol.

FIG. 42DD shows C2C12 cells that were treated with 100 ng/ml of Wnt3a,BMP2 (100 ng/ml) or combined BMP2 with Dkk1 at indicated time. Resultsare shown as mean±SD (n=3) and representative of three independentexperiments. **P<0.01 and ****P<0.0001 versus control.

FIGS. 43A-43LL show that myeloma-derived DKK-1 disrupts Wnt-regulatedosteoprotegerin and RANKL production by osteoblasts.

FIGS. 43A-43D show that Wnt3a induced increase in OPG mRNA and proteinin osteoblast progenitor cells. C2C12 cells (FIGS. 43A and 43C) andSaos-2 cells (FIGS. 43B and 43D) were treated with serial concentrationsof recombinant Wnt3a for indicated times. The OPG mRNA (FIGS. 43A and43B) was amplified by qPCR analysis. The supernatant of treated cells(FIGS. 43C and 43D) was harvested and subjected to ELISA for measurementof OPG protein. Protein in lysate (1 mg) was subjected to theGST-E-cadherin assay. Following SDS-PAGE analysis, uncomplexedbeta-catenin was detected by anti-beta-catenin antibody (FIGS. 43C and43D). The results are a mean±SD (n=4). Results are representative ofthree independent experiments. * P<0.05 versus control.

FIGS. 43E-43J show that DKK-1 inhibition of Wnt3a induced OPG mRNA andprotein in osteoblast cells. C2C12 (FIG. 43E) and Saos-2 (FIG. 43F)cells were stimulated with or without Wnt3a after prior treatment withrecombinant DKK-1 with indicated concentrations and then lysed. 0.5 mgof protein from cell lysates was subjected to the GST-E-cadherin assay.Following SDS-PAGE, uncomplexed b-catenin was detected by anti-b-cateninantibody. Total RNA was isolated from treated C2C12 (FIG. 43G) andSaos-2 (FIG. 43H) cells and OPG mRNA quantified. The supernatant ofC2C12 (FIG. 43I) and Saos-2 (FIG. 43J) cells treated, as above for 72hours, was harvested and subjected to ELISA for measurement of OPG. Theresults are shown as mean±SD (n=3). Results are representative of threeindependent experiments. ** P<0.01, *** P<0.001, **** p<0.00001 versuscontrol.

FIGS. 43K-43Q show that ectopic expression of DKK1 diminished Wnt3ainduced OPG mRNA and protein in osteoblast cells. The expression of DKKfamily members in C2C12 (FIG. 43K) and human osteoblast cell lines (FIG.43L) as determined by RT-PCR analysis are presented. Concentration ofDKK1 protein in culture supernatant of indicated cell lines by ELISAanalysis (FIG. 43M). C2C12 cells were stable transfected with an emptyvector or DKK1-expressing vector. DKK1 protein expression was detectedby the anti-V5 antibody (FIG. 43N) and the concentration of DKK1 proteinis shown in FIG. 43O. The cells were treated with recombinant 100 ng/mlof rWnt3a. Relative OPG mRNA (FIG. 43P) and OPG protein concentration(FIG. 43Q) was measured by qPCR or ELISA analysis as described in FIG.43A-43D. Data represent the mean±SD (n=3) of representative experiments.*P<0.05, ***p<0.001, and **** p<0.00001 versus control.

FIGS. 43R-43V show that knockdown of DKK1 by shRNA restored Wnt3ainduced OPG in Osteoblast. C2C12 cells were transiently infected withsupernatant containing control siRNA (shCont) or shRNA specific for DKK1for indicated times. Total RNA was then isolated and subjected to RT-PCRfor detecting DKK1 mRNA (FIG. 43R). cDNA from 24 hours was subject toqPCR to confirm DKK1 mRNA expression (FIG. 43S). Supernatants of thecells were harvested and subjected to ELISA for measuring DKK1 protein(FIG. 43T). The infected cells were treated with rWnt3a for 48 hours andRNA and supernatants were harvested and subjected to qPCR and ELISAanalysis for OPG mRNA (FIG. 43U) and protein (FIG. 43V). Data representthe mean±SD (n=3) of representative experiments. **P<0.01, ***p<0.001,versus control.

FIGS. 43W-43BB show that co-culturing osteoblast cells with DKK1expressing MM cells inhibits Wnt3a-induced OPG. A MM cell line, OPM-2was transfected with pEF6 vector (pEF/EV) or pEF6/DKK-1. DKK1 protein inpEF/EV or pEF/DKK1 was determined (FIG. 43W). The concentration of DKK1protein in culture supernatants in pEF/EV and pEF/DKK1 was measured byELISA (FIG. 43X). C2C12 cells were co-cultured with pEF/EV or pEF/DKK1cells in presence rWnt3a or control for the indicted times. OPGsynthesis in these cells, as measured by qPCR, is presented (FIG. 43Y).Supernatants of the C2C12 were harvested and subjected to ELISA analysisto measure OPG protein concentration (FIG. 43Z). The results are shownas mean±SD (n=4). Results are representative of three independentexperiments. ** P<0.01, *** P<0.001, **** p<0.00001 versus control.C2C12 cells were cultured with primary CD1380-positive plasma cells fromtwo MM pateins (P#1 and P#2) for 48 hours in the presence or absence ofrWnt3a for 48 hours. The OPG mRNA in C2C12 cells was determined by qPCR(FIG. 43AA). OPG protein in supernatant of the cultures was measured byELISA (FIG. 43BB).

FIGS. 43CC-43FF show that neutralization of DKK1 rescues OPG expressionin osteoblasts grown in the presence of MM sera or primary MM cells.C2C12 cells were treated with rWnt3a or vehicle after prior treatmentwith bone marrow sera from MM patients (n=8) containing low (L) (2.7 to8.5 ng/ml) or high (H) (104.5 to 273.5 ng/ml) concentration of DKK1 orrecombinant DKK1 (100 ng/ml) as positive control for 48 hours. The cellswere treated with rWnt3a or control vehicle after prior treatment with25% sera from MM patients (n=21) containing mouse Ig or anti-DKK1antibody for 48 hrs. C2C12 cells were co-cultured with CD138 positiveplasma cells from MM in the presence or absence of Wnt3a, control IgG oranti-DKK1 antibody. OPG mRNA was determined by qPCR from the RNA, andOPG protein measured by ELISA of cell culture supernatants (FIGS.43CC-43FF). ** P<0.01, *** P<0.001, **** p<0.00001 versus control.

FIGS. 43GG-43LL shows that DKK1 and sera from MM patients inhibitsWnt3a-induced suppression of RANKL in osteoblast. C2C12 (FIG. 43GG),Saos-2 (FIG. 43HH) and MG63 (FIG. 43II) cells were treated with Wnt3a-CMor Cont-CM after prior treatment with 100 ng/ml of DKK1 protein for 48hours. RANKL mRNA was analyzed by qPCR. C2C12 cells transfected withempty vector (pEF/EV) or the vector carrying DKK1 cDNA (pEF/DKK1) werecultured in the presence of 100 ng/ml of BMP-2 and presence or absenceof rWnt3a protein (100 ng/ml). The RNA and supernatant were harvestedand subjected to (FIG. 43JJ) qPCR of RANKL mRNA (FIG. 43KK) or ELISA forRANKL protein (FIG. 43LL). C2C12 cells were treated with Wnt3a proteinafter prior incubation with sera from MM patients (n=8) containing lower(<10 ng/ml) or higher concentration of DKK1 (>100 ng/ml) for 48 hours(FIG. 43LL). RANKL mRNA was analyzed by qPCR. The results are shown asmean±SD (n=3). * P<0.01, ** P<0.001, versus control.

FIG. 44 shows the SCID-rab model for primary myeloma. A small piece ofrabbit bone was implanted subcutaneously in SCID mice. Myeloma cellsfrom different patients were injected directly into the implanted bone.Myeloma cells from more than 85% of patients were successfully engraftedin this model.

FIG. 45 shows that anti-DKK1 treatment is associated with an increasednumber of osteoblasts and a reduced number of osteoclasts in myelomatousbone of SCID-rab mice. Bone sections were stained for TRAP to identifyosteoclasts and for osteocalcin to identify osteoblasts.

FIG. 46 shows that anti-DKK1 treatment increases osteoblast activity andreduces osteoclast numbers in myelomatous SCID-rab mice. Sequentialsections were stained for TRAP and osteocalcin. Note that whereascontrol bone had increased osteoclast numbers and diminishedosteoblasts, anti-DKK1 treatment resulted in increased osteoblastnumbers and reduced those of the osteoclasts.

FIG. 47 shows that anti-DKK1 increases bone marrow density (BMD) andinhibits myeloma growth in myeloma-bearing SCID-rab mice. Myelomatousrabbit bone marrow density and circulating human immunoglobulins (hIg)were measured before treatment and at the end of the experiment.

FIG. 48 shows that blocking DKK1 increases bone mass in myelomatousSCID-rab mice. X-ray radiographs of the implanted rabbit bone of controland anti-DKK1-treated mice, before initiation of treatment (Pre-Rx) andat the end of the experiment (final) are shown. Many lytic lesions wereevident in both mice at Pre-Rx. However, although the bone losscontinued to increase in the control mouse, anti-DKK1 treatment resultedin increased bone mass and partial repair of lytic lesions.

FIG. 49 shows that increased osteoblast activity is associated withreduced tumor burden in SCID-rab mice treated with anti-DKK1.Myelomatous bone section from control and anti-DKK1-treated mice wereimmunohistochemically stained for osteocalcin. The bone treated withanti-DKK1 but not the control antibody was associated with remarkableincrease in osteoblast (OB) number (stained brown). This area designatedas OB zone was depleted of viable myeloma cells.

FIGS. 50A-50C show that DKK1 neutralizing antibody promotes boneformation in nonmyelomatous bones. FIGS. 50A-50B demonstrates changes inbone marrow density of the implanted bones (FIG. 50A) and mouse femur(FIG. 50B) in mice treated with control IgG and anti-DKK1 neutralizingantibody. FIG. 50C shows changes in bone marrow density of theuninvolved mouse femur in myelomatous hosts treated with the control IgGand anti-DKK1 neutralizing antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention demonstrates that the secreted WNT signalingantagonists DKK-1 and FRZB mediate bone destruction seen in multiplemyeloma. These data strongly implicate these factors in causingosteoblast anergy and contributing to multiple myeloma bone disease bysuppressing the normal compensatory bone production that follows boneloss.

The role of multiple myeloma plasma cells in stimulating osteoclastactivity has been intensely investigated and several key linksestablished. Data presented herein provide for the first time evidenceof a possible mechanistic explanation of osteoblast dysfunction inmultiple myeloma. These are significant observations in that inhibitionof WNT signaling causes defects in osteoblast function. The secretedDKK-1 and FRZB could account for both the systemic osteoporosis seen inmultiple myeloma as well as the exaggerated local bone destructionproximal to plasma cells foci.

Importantly, DKK-1 and FRZB act to inhibit WNT signaling throughindependent mechanisms, indicating that their co-expression may havesynergistic effects. Thus, these genes could be used to predict extentof bone disease and future risk of developing bone disease. Moreover,inhibitors of these proteins could be used to block bone disease. It isalso possible that these factors play a role in osteoporosis in thegeneral population.

WNT Signaling Pathway

Wnt genes comprise a large family of secreted polypeptides that areexpressed in spatially and tissue-restricted patterns during vertebrateembryonic development. Mutational analysis in mice has shown theimportance of Wnts in controlling diverse developmental processes suchas patterning of the body axis, central nervous system and limbs, andthe regulation of inductive events during organogenesis. The Wnt familyof secreted growth factors initiates signaling via the Frizzled (Fz)receptor and its coreceptor, LDL receptor-related protein 5 or 6 (LPR5or LRP6), presumably through Fz-LPR5/LRP6 complex formation induced byWnt.

Secreted antagonists of Wnt include Frizzled (Fz)-related proteins(FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (DKK).Frizzled (Fz)-related proteins, Cerberus and Wnt inhibitory factor haveall been shown to act by binding and sequestering Wnt. Unlike Wntantagonists which exert their effects by molecular mimicry of Fz or Wntsequestration through other mechanisms, Dickkopf-1 (DKK-1) specificallyinhibits canonical Wnt signalling by binding to the LPR5/LRP6 componentof the receptor complex.

DKK-1 is a head inducer secreted from the vertebrate head organizer andinduces anterior development by antagonizing Wnt signaling. DKK-1 is ahigh-affinity ligand for LRP6 and inhibits Wnt signaling by preventingFz-LRP6 complex formation induced by Wnt. DKK-1 binds neither Wnt norFz, nor does it affect Wnt-Fz interaction. DKK-1 function in headinduction and Wnt signaling inhibition strictly correlates with itsability to bind LPR5/LRP6 and to disrupt the Fz-LPR5/LRP6 association.LPR5/LRP6 function and DKK-1 inhibition appear to be specific for theWnt/Fz beta-catenin pathway. These findings thus reveal a novelmechanism for Wnt signal modulation.

WNT Signaling and Osteoblast Differentiation

Recent studies have shown that the Wnt signaling pathway is critical forosteoblast differentiation and function. Mice with a targeted disruptionin the gene for low-density lipoprotein receptor-related protein 5(LRP5) developed a low bone mass phenotype. LRP5 is expressed inosteoblasts and is required for optimal Wnt signaling in osteoblasts. Invivo and in vitro analyses indicated that this phenotype becomes evidentpostnatally, and it was secondary to decreased osteoblast proliferationand function in a Cbfa1-independent manner. In humans, mutations in LRP5cause the autosomal recessive disorder osteoporosis-pseudogliomasyndrome (OPPG). Osteoporosis-pseudoglioma syndrome carriers havereduced bone mass when compared to age- and gender-matched controls.

Importantly, separate and distinct mutations in LRP result in a highbone mass phenotype. In contrast to the osteopororsis-psuedogliomamutations, the high bone mass traits are gain of function mutations.Markers of bone resorption were normal in the affected subjects, whereasmarkers of bone formation such as osteocalcin were markedly elevated.Levels of fibronectin, a known target of signaling by Wnt, were alsoelevated. In vitro studies showed that the normal inhibition of Wntsignaling by Dickkopf-1 (DKK-1) was defective in the presence of themutation and that this resulted in increased signaling due to unopposedWnt activity. These findings demonstrated the role of altered LRP5function in high bone mass and point to DKK as a potential target forthe prevention or treatment of osteoporosis.

WNT Signaling and Bone Disease in Multiple Myeloma

Indirect evidence of a role of DKK-1 in osteoblast function has beenprovided by identification of gain of function mutations in LRP-5 beinglinked to a high bone mass phenotype. In addition, targeted disruptionof secreted firzzled-related protein (SFRP-1), a homologue of FRZB(SFRP-3), leads to decreased osteoblast and osteocyte apoptosis andincreased trabecular bone formation.

A quantitative trait loci (QTL) influencing bone mass has been localizedto the LRP-5 region, suggesting that the population at large havedifferent risk of developing osteoporosis. It is conceivable thatmultiple myeloma bone disease may be influenced by the combined effectsof DKK-1/FRZB expression with an inherited predisposition to low bonemass conferred by inherited LRP-5 alleles. Multiple myeloma cases may begenotyped for LRP-5 allele variations and correlate this informationwith bone disease, and DKK-1 and FRZB expression.

Monoclonal gammopathy of undetermined significance (MGUS), a plasma celldyscrasia that is predisposed to develop into multiple myeloma, isdifferentiated from multiple myeloma by the lack of obvious bonedisease. The significance of discovering DKK-1 and/or FRZB expression ina third of monoclonal gammopathy of undetermined significance is unclearbut could suggest that these cases may be at higher risk for developingmultiple myeloma. As with multiple myeloma, this predisposition may alsobe related to inherited LRP5 alleles. Alternatively, these monoclonalgammopathy of undetermined significance cases could have underlyingpreclinical bone disease that is not yet apparent by radiological scans.

Data presented herein suggests a model for how DKK-1 expression bymultiple myeloma plasma cells can be linked to multiple myeloma diseasegrowth control and bone destruction and how these two phenomena can beintegrated by one molecule. In the model, primary multiple myelomaexpress high levels of DKK and these levels can be increased with drugtherapies used to treat the disease. High levels of DKK-1 likely induceapoptosis of multiple myeloma cells and could explain the relativelyslow progression of the disease in its early phase as cell growth istempered by high rate of DKK-1 induced apoptosis. However, as thedisease progresses there is an osteoclast-induced reduction in JUN andDKK-1 that eventually develops into a constitutive loss of JUN and DKK-1expression as seen in extramedullary disease.

Thus, if one were to view DKK-1 expression from the perspective of themultiple myeloma plasma cells, high levels of DKK-1 expression could beseen as positive feature of the disease. However, with the mesenchymalcell lineage being exquisitely sensitive to DKK-1 induced apoptosis, thehigh levels of this secreted product likely has a double edge to it inthat it also induces massive programmed cell death of osteoblastprecursors and possibly even mesenchymal stem cells. It is expected thathigh levels of DKK-1 early in the disease could lead to a permanent lossof mesenchymal stem cells, a notion supported by the observed lack ofbone repair after remission induction or during disease progression whenosteoclasts likely suppress DKK-1 secretion by multiple myeloma plasmacells. Thus, exploitation of this knowledge might lead to thedevelopment of new therapies for multiple myeloma that accentuateDKK-1's effects on multiple myeloma plasma cells, but at the same timeprevent DKK's bone damaging effects on osteoblast or their precursors.

The present invention also describes a molecular mechanism by which DKK1likely inhibits osteoblast differentiation and contributes to myelomabone disease. Initial experiments (FIGS. 42A and 42B) demonstrated thatmouse and human pluripotent mesenchymal cell lines are capable oftransducing a canonical Wnt signal. These cells expressed mRNAcorresponding to multiple Wnt receptors and the LRP5/6 co-receptors,suggesting pre-osteoblasts are capable of interacting with Wnt ligand.Treatment with exogenous Wnt3a led to enhanced activation of afunctional signaling pathway as evidenced by increases of bothuncomplexed and transcriptionally active forms of b-catenin in thecytosol. Accumulation of beta-catenin in the cytoplasm leads to nucleartranslocation and binding of TCF/LEF family members to form complexescapable of activating transcription. RT-PCR analysis (FIG. 42B) revealedthat all members of the TCF family are expressed in mouse C2C12 cells,and TCF1, 4, and LEF1 are expressed in human osteoblast-like cell lines.Moreover, functional activation of these transcription factors followingWnt3a treatment was demonstrated using a luciferase reporter construct.

It has been reported that exogenous Dkk1 blocks Wnt3a-inducedstabilization of b-catenin and inhibits activation of the canonical Wntpathway in MM cells (qiang and Rudikoff, 2004; Qiang et al., 2003). Inthe present invention (FIG. 42C), it was observed that Dkk1 cansimilarly regulate Wnt-induced stabilization of beta-catenin in mouseand human pre-osteoblasts as Dkk1 protein produced by MM cell linesstably expressing the protein, or MM patient bone marrow plasmacontaining high levels of Dkk1, inhibited stabilization of b-catenin byWnt3a. It is also noted that Dkk1 completely attenuates Wnt3a-inducedTCF/LEF transcriptional activity in mouse pre-osteoblasts. Conflictingresults exist as to the role of Dkk2 and other Dkk family members onpre-osteoblast differentiation (Li et al., 2005; van der Horst et al.,2005). Analysis of mRNA expression in the tested cell lines revealedrelative expression of Dkk family mRNAs in the following order:Dkk3>Dkk2>Dkk1=Dkk4. Treatment of pre-osteoblast cells with supernatantscontaining Dkk2 inhibited b-catenin stabilization similarly to thatobserved with Dkk1. In addition, over-expression of Dkk1 or Dkk2directly in C2C12 cells reduced endogenous b-catenin levels.

Although osteoblasts clearly respond to Wnt3a by enhanced activation ofthe canonical Wnt/b-catenin pathway, Wnt3a alone had no apparent effecton differentiation of osteoblast precursors as reflected by ALPproduction. Surprisingly, Dkk1 (or Dkk2) significantly blockedBMP-2-induced differentiation (FIG. 42D). These results indicate that anautocrine canonical Wnt signal present in OB precursor cells isnecessary for BMP-2-induced differentiation. This conclusion issupported by several additional observations. First, abundant expressionof multiple Wnt mRNAs was observed in all cell lines (unpublished data),in addition to Fz and LRP5/6 receptors (FIG. 42A). Second, silencing ofLRP5 or LRP6 mRNA expression completely abrogated BMP-2-induced ALPactivity (FIG. 42F). This result is consistent with previous reportsshowing that lack of LRP5 (Kato et al., 2002) and LRP6 (Kokubu et al.,2004) reduced bone formation and osteoblast differentiation in a mousemodel. Third, higher levels of uncomplexed and transcriptionally activebeta-catenin protein exist in these cells relative to osteoclasts, MMcells and other cell types. Fourth, blockage of endogenous b-catenin bya dominant negative b-catenin construct significantly inhibitedBMP-2-induced C2C12 differentiation (FIG. 42E) in agreement with dataindicating that over-expression of active b-catenin increased bone mass(Glass et al., 2005). Finally, constitutive expression of Dkk1 (or Dkk2,not shown) led to, not only reduced endogenous levels of uncomplexed andtranscriptionally active b-catenin, but also decreased ALP activityfollowing BMP-2 treatment. These results are consistent with previous invivo studies showing that deletion of Dkk1 leads to increased boneformation and bone mass (Morvan et al., 2006; van der Horst, 2005), andover-expression of Dkk1 in transgenic mice results in osteopenia (Li etal., 2006). The present study provides direct evidence that endogenousb-catenin levels in mesenchymal cells are necessary for osteoblastdifferentiation, and Dkk1 from MM cells inhibits this process.

Since Dkk1 can block BMP-2 induced ALP production and Wnt signaling isnecessary to induce differentiation, the possibility exists for crossregulation between these two pathways. Experiments to test thishypothesis revealed that BMP-2 treatment alone did not induce increasedlevels of beta-catenin over steady state (FIG. 42G), nor did it affectTCF/LEF transcriptional activity, nor did Wnt3a increase ALP activity(FIG. 42D). Furthermore, no association was observed between Dkk1 andBMP-2 receptors, and neither Wnt3a nor Dkk1 treatment led to activationof Smad-1, -5, and -8 (important downstream targets of BMP-2 activationthat play a pivotal role in BMP-2-induced mesenchymal celldifferentiation) (Canalis et al., 2003). Moreover, Cbfa-1/Runx2transcription factor activity and increase in Smad6 mRNA induced by BMPtreatment did not change in response to Wnt or DKK1, further indicatingthat Wnt-signaling does not activate the BMP pathway. However, the factthat Dkk1 did inhibit the canonical Wnt pathway and also BMP-2-inducedosteoblast differentiation in the present study suggests that there isindeed co-regulation between BMP-2 and Wnt signaling of osteoblastdifferentiation. The nature of this co-regulation is the focus of activeinvestigation.

In contrast to the lack of cross regulation described above, otherstudies have suggested that BMP-2 increases endogenous Wnt mRNAexpression to promote increased ALP activity (Rawadi et al., 2003; Chenet al., 2006). Furthermore, BMP-2 and a b-catenin mutant withconstitutive transcriptional activity (DeltaN151) synergized tostimulate ALP activity, osteocalcin gene expression, and matrixmineralization (Mhalaviele et al., 2005). However, in the presentexperiments, BMP-2 did not induce increased stabilization of cytosolic,free b-catenin in mouse and human pre-osteoblast cells, nor BMP-2 aloneis able induced TCF/LEF transcriptal activity, nor did BMP-2synergizesWnt3a-ainduced TCF/LEF activity indicating that, under these conditions,BMP-2 is unlikely to alter baseline or steady state levels of Wntsignaling sufficient, and required, for BMP-2 induced differentiation.It appears that cross talk between BMP-2 and a canonical Wnt pathwaydoes not occur at, or above, the analyzed downstream targets of eachsignaling pathway. Consistent with this hypothesis, Nakashima andcolleagues have previously reported that BMP-2 alone failed to increaseTCF/LEF activity (Nakashima et al., 2005) although these two pathwaysare required for preosteoblast differentiation. Moreover, Mbalaviele andcolleagues provided in vivo evidence that BMP-2 does not influenceTCF/LEF activity related to that activated by b-catenin mutant (ΔN151)in C3H10T1/2 cells (Mhalaviele et al., 2005). However, the possibilitythat interactions between Wnt and BMP-2 signaling pathways occur throughalternate cascades cannot be excluded. In fact, in other systems, it hasbeen reported that beta-catenin and LEF/TCF form complexes with Smads(Hu and Rosenblum, 2005). Additionally, Wnt3a and BMP-2 (Willert et al.,2002) can induce expression of the ID2 gene and both induced MSX1 geneexpression (Binato et al., 2006). Further studies will be required toclarify these differences.

Since it is thought that LRP5 or LRP6 can act redundantly, the reasonsfor observing an almost complete loss of BMP-2 induced ALP activity,when only one of the two was silenced are not clear. It may be that theamount of LRP5 or LRP6 on cell surface tightly regulates Wnt signalingrequired for BMP-2 induced ALP. While required, either alone is notsufficient. Indeed, Wnt-1 induced TCF/LEF transcriptional activity wasalmost completed blocked in fibroblast in LRP6 null mice in the presenceof LRP5 (Kokubu et al., 2004). On the other hand, expression ofloss-function of LRP5 mutant almost completely abolishes BMP-2 inducedALP activity in the presence of LRP6 in ST2 pluripotent bone marrowstromal cells (Gong et al., 2001). Another explanation could be that thetrafficking of LRP5 and LRP6 on the cell surface might regulate Wntsignaling. This is consistent with recent studies that show thatR-Spondin-1 regulates LRP6 cell surface levels of LRP6 by interferingwith Dkk1/Kremen-mediated internalization of LRP6 (Binnerts et al.,2007). Further studies will be needed to distinguish these hypotheses

In conclusion the above studies have revealed that autocrine Wntsignaling in osteoblasts is necessary to promote BMP-2-mediateddifferentiation of pre-osteoblast cells, while Wnt signaling alone isnot capable of inducing such differentiation. Dkk1 inhibits this processand may be a key factor regulating pre-osteoblast differentiation,thereby emphasizing the importance of Dkk1 as a molecular target fornovel therapeutic approaches to modulate myeloma bone disease.

The present invention also demonstrates that DKK1 may contribute toosteolytic bone lesion in MM by attenuating Wnt signaling in osteoblaststhat prevents their differentiation and hence alters the expression ofOPG and RANKL in favor of RANKL, which in turn leads to increasedosteoclastogenesis in the local environment surrounding the plasma cellfoci within the bone. The evidence supporting this model are thefollowing: 1) DKK1 inhibits Wnt3a-induced stabilization of beta-cateninand reduces free-beta-catenin in both mouse and human osteoblast cells,2) exogenous administration of DKK1 or constitutive expression of DKK1dramatically diminished Wnt3a induced OPG expression in osteoblasts, 3)silencing DKK1 expression in human osteoblast-like cells expressingendogenous DKK1 increases sensitivity and reaction to Wnt3a stimulationas determined by increases in OPG expression, 4) MM bone marrow serumcontaining high DKK1 blocked Wnt3-mediated OPG expression, 5) mimickingthe interaction between osteoblasts and MM cells in the bone marrow, aco-culture system also revealed that the DKK1-secreting OPM-2 mM cellline and primary CD138-selected plasma cells from MM patientsdramatically attenuated Wnt3a-induced OPG mRNA and protein production byosteoblasts, and 6) a neutralizing DKK1-antibody could restore OPGexpression in osteoblasts that was inhibited by the presence of MM bonemarrow serum or primary MM plasma cells. Taken together, these resultssupport the notion that DKK1 interrupts Wnt signaling-regulated boneresorption through regulation of osteoclastogenesis by inhibiting OPGexpression. Indeed, OPG levels are decreased in myeloma serum relativeto healthy controls (Lipton et al., 2002; Seidel et al., 2001). Theimportance of OPG is evidenced by the fact that administration ofrecombinant OPG or OPG peptidomimetic, OP34, can inhibit bone resorptionand MM-associated osteolytic bone disease in murine models (Vanderkerkenet al., 2003; Heath et al., 2007). In fact, Wnt signaling appears toindirectly inhibit osteoclastogenesis as well. It was observed thatsupernatants from osteoblast cells transfected with domain negativebeta-catenin contain higher RANKL and lower OPG levels and thesesupernatants increase human osteoclasts from CD34 mononuclear cellsisolated from bone marrow of MM patients relative to control supernatant(Ya-Wei Qiang unpublished data, 2007). This is consistent with in-vivodata that show that deletion of beta-catenin results in marked increasein osteoclast cell number (Holmen et al., 2005).

In contrast to the inhibitory effect of DKK1 on Wnt-stimulated OPGexpression in osteoblast cells interacting with MM cells, DKK1 restoresRANKL expression in osteoblast cells. Supporting this hypothesis are thefollowing observations: 1) DKK1 significantly reversed Wnt3a-mediateddownregluation of RANKL expression in mouse and human osteoblast-likecell lines, and 2) overexpression of DKK1 in osteoblast cells and MMserum with high DKK1 levels reversed Wnt3a-mediated downregulation ofRANKL expression in mouse and human osteoblast-like cell lines. Theseresults are consistent with studies in which DKK1 increases RANKLexpression in the mouse osteoblast cell line C3H10T1/2. A role of Wntsignaling in the regulation of RANKL expression was first recognized byHolmen and colleagues who reported that an increase in canonical Wntsignaling by deletion of the Wnt inhibitory molecule APC results in anincrease in RANKL expression in normal osteoblast cells in mice (Holmenet al., 2005). More recently, Spencer and colleagues illustrated thatthe human RANKL promoter contains TCF/LEF binding sites andoverexpression of full-length beta-catenin inhibits RANKL promoteractivity through a currently unknown mechanism in MC3T3-E1 cells(Spencer et al., 2006). Although the source of RANKL is controversial,several groups have reported a role for RANKL in MM-triggered bonelesions. RANKL is upregulated in myeloma cells (Giuliani et al., 2001;Pearse et al., 2001) and increased levels of RANKL in MM serum is usedas prognostic index for indicating a survival in MM patients (Terpos etal., 2003).

To reach comparable levels of beta-catenin stabilization, higherconcentrations of Wnt3a were required in human osteoblasts than mouseosteoblasts, which may be attributable to dramatically higher levels(approximately 50-fold) of endogenous DKK1 in human osteoblast lines,since mouse and human lines have similar expression patterns ofendogenous Wnt ligands and LRP5/6 co-receptor and Fz receptors.Consequently, ectopic constitutive expression of DKK1 in mouse C2C12cells, which lack DKK1 expression, blocked Wnt3a-induced OPG expressionto an extent similar to that seen with human osteoblast cells, whichexpress high levels of endogenous DKK1. In contrast, knockdown ofendogenous DKK1 expression in human osteoblast cells restoredsensitivity to Wnt3a stimulation as exhibited by an increase in OPGexpression. Thus, endogenous DKK1 in osteoblasts appears to be a keyfactor determining sensitivity to exogenous Wnt stimulation. Thedifference in DKK1 expression between these cells might represent thedifferent specific stage of osteoblast differentiation that the cellsrepresent, as the mouse osteoblast progenitor cell line C2C12 representsmore immature progenitor cell the human osteosarcoma cells used(Katagiri et al., 1994). This notion is supported by the fact that DKK1expression is high in late-stage osteoblast cell line KS463 (van derHorst et al., 2005). One can not exclude the possibility that thisdifference might reflect differences between mouse and man as human bonemarrow derived mesenchymal cells express high levels of DKK1 (Giulianiet al., 2007) and DKK1 regulates human, but not mouse, mesenchymal celldifferentiation into adipocytes or osteoblasts. Hence, the endogenousDKK1 levels in osteoblast cells should be considered an important factorwhen selecting as a model for studies role of Wnt signaling inregulation of OPG and RANKL

Although Wnt3a regulates both OPG and RANKL expression and DKK1interrupts this process, it is interesting to note that Wnt3astimulation had stronger effects on OPG expression than that of RANKL inthese experiments. Wnt3a induced a much higher increase in OPGexpression in response to Wnt3a compared with the inhibitory effect onRANKL expression. In addition, while anti-DKK1 antibody restoredDKK1-suppressed OPG expression, it had no effect on DKK1-mediatedincrease of RANKL in osteoblast cells in coculture with primary MMcells. Thus, OPG seems to be more sensitive to Wnt signaling than RANKL.However, it has clearly been shown that overexpression of DKK1 andblockage of endogenous canonical Wnt signaling by expression of dominantnegative b-catenin significantly increases RANKL mRNA and protein.

Thus, it is likely that DKK1-mediated suppression of OPG, rather thanits effect to release a block to RANKL expression, may be the moreimportant event contributing to MM OBL. However, the possibility thatendogenous Wnt ligands regulate OPG and RANKL and as such regulatehomeostasis of osteoclastogenesis in normal physiological conditionscannot be excluded since osteoblast cells express many Wnt ligands.Another possibility that was not addressed herein was whether endogenousWnt signaling modulates RANKL expression at levels that are bellow thelevels of sensitivity of current methods used to detect RANKL protein.This is supported by the fact that constitutive expression of DKK1 andlack of transcriptional activity of beta-catenin in osteoblast cellsrestores RANKL expression.

It is noteworthy that Gunn and colleagues have shown that conditionedmedia from MSCs can induce multiple myeloma cells lines to produce DKK1and that these cells also produce high levels of IL-6 (Gregory et al.,2003; Gunn et al., 2006) a myeloma growth factor (Kishimoto, 2005).Importantly, Gunn et al showed that IL-6-dependent myeloma cell linesgrowth in MSC conditioned media and that this growth is inhibited when aneutralizing antibody to IL-6 is added to the cultures.

Furthermore, the present invention also demonstrated that blocking ofDKK1 activity in primary human myeloma-bearing SCID-rab mice wasassociated with increased osteoblast numbers and reduced osteoclastactivity. This decreased osteoclast numbers in myelomatous bones fromSCID-Hu mice could be due to a reduction of RANKL and increase in OPG.These effects resulted in prevention of bone resorption, increased boneformation and most importantly inhibition of tumor burden. The presentinvention also establishes, that Multiple Myeloma bone disease and tumorgrowth are interdependent, as blocking DKK1 activity, was accompanied byinhibition of Multiple Myeloma by blocking DKK1 activity progression.These in vivo data confirmed that DKK1 is critical factor involved inmyeloma bone disease and tumor progression. Thus, therapeutic approachesto inhibit DKK1 activity in patients with myeloma will not only improveskeletal complications and quality of life but also help controlmyeloma. In addition, the present invention also demonstrated, thatblocking of DKK1 activity in SCID-rab mice had bone anabolic effects onnon-myelomatous bones, suggesting that DKK1 neutralization may havebroad applications in bone disorders.

Taken together, the present invention proposes a working hypothesis thatmyeloma-derived DKK1 can act as a master regulator of OBL and myelomadisease survival. DKK1-mediated inhibition of Wnt-regulated osteoblastdifferention results in a loss of their functional activity to replacebone resorbed by osteoclasts. This leads to increased expression ofIL-6, an essential survival factor for myeloma. This block of Wntsignaling also leads to a loss of expression of OPG and increasedexpression of RANKL. It is contemplated that the shift in theRANKL-to-OPG ratios, at the site of boney plasmacytomas, beingpropagated by high local concentrations of IL-6, results in increasedlocal osteoclastogenesis and increased bone resorption with no anabolicresponse. Thus, DKK1 represents an important new and therapeuticallytractable target as has been suggested herein and by preclinicalstudies.

In one embodiment of the present invention, there is provided a methodof controlling bone loss in an individual, comprising the step ofinhibiting a WNT signaling antagonist at the nucleic acid or proteinlevel. Specifically, the inhibition of Wnt signaling antagonist mayblock induction of Wnt ligand, restore RANKL/OPG levels or both.Examples of WNT signaling antagonist may include but are not limited tosoluble frizzled related protein 3 (SFRP-3/FRZB) or the human homologueof Dickkopf-1 (DKK1). The inhibition at the nucleic acid level may bedue to Wnt antagonist specific peptide nucleic acid or siRNA.Alternatively, the inhibition at the protein level may be due to saidWnt antagonist specific antibodies, anti-sense oligonucleotides or smallmolecule inhibitors. Examples of individual who may benefit from such amethod may include but are not limited to ones with multiple myeloma,osteoporosis, post-menopausal osteoporosis and malignancy-related boneloss. The malignancy-related bone loss may be caused by breast cancermetastasis to the bone or prostate cancer metastasis to the bone.

In another embodiment of the present invention, there is a method oftreating bone disease in an individual, comprising the step of:administering to the individual a pharmacologicallly effective amount ofan inhibitor of a WNT signaling antagonist such that the administrationblocks induction of Wnt ligand, restores RANKL/OPG levels or both.Examples of the WNT signaling antagonist may include but are not limitedto soluble frizzled related protein 3 (SFRP-3/FRZB) or the humanhomologue of Dickkopf-1 (DKK1). The inhibitor may inhibit the Wntsignaling antagonist at the nucleic acid or protein level. Examples ofthe inhibitor at the nucleic acid level and protein level and thoseindividuals benefiting from such a method are same as discussed supra.Additionally, the inhibitor may treat the bone disease by preventingbone resorption, increasing bone formation or both.

In yet another embodiment of the present invention, there is a method ofinhibiting tumor growth in bone of an individual, the method comprisingthe step of blocking the activity of DKK1. Generally, the DKK1 activityis blocked by administering anti-DKK1 antibodies, DKK1 anti-senseoligonucleotides or small molecule inhibitor to the individual.Moreover, an individual who will benefit from such a method although notlimited to includes one who has multiple myeloma, metastatic breastcancer or prostate cancer.

In another embodiment of the present invention, there is a method ofscreening for a compound that controls bone loss and inhibits humanmyeloma cell growth, comprising: engrafting human myeloma cells in arabbit bone implanted in a SCID-rab mouse, administering the compound tothe mouse; and comparing bone mineral density of the implanted bone andlevel of serum human monoclonal immunoglobulin in the mouse with acontrol SCID-rab mouse that has not received the compound, where anincrease in the bone mineral density and a decrease in the level ofserum immunoglobulin in the treated mouse compared to the control mouseindicates that the compound controls bone loss and inhibits humanmyeloma growth. Generally, the compound is an inhibitor of WNT signalingantagonist. Specifically, the WNT signaling antagonist is humanhomologue of Dickkopf-1 (DKK1) or soluble frizzled related protein 3(SFRP-3/FRZB).

In yet another embodiment of the present invention, there is a method ofinhibiting multiple myeloma growth in an individual suffering frommultiple myeloma, said method comprising the step of blocking theactivity of DKK1. This method may further comprise increasingosteoblastogenesis and decreasing osteoclastogenesis. The increase inosteoblastogenesis and the decrease in osteoclastogenesis is due toblocking of induction of Wnt ligand, restoring RANKL/OPG levels or both.Examples of inhibitors blocking the DKK1 activity are the same asdiscussed supra.

As used herein, the term, “a” or “an” may mean one or more. As usedherein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” or “other” may mean at least a second or more ofthe same or different claim element or components thereof. s discussedherein, the inhibitor of Wnt antagonist described herein may be used invitro or ex vivo by exposing the cell culture to the composition in asuitable medium. In vivo may be achieved by any known methods in theart.

The inhibitor of Wnt antagonist described herein or known in the art maybe administered independently one or more times to achieve, maintain orimprove upon a therapeutic effect. It is well within the skill of anartisan to determine dosage or whether a suitable dosage of such aninhibitor comprises a single administered dose or multiple administereddoses. An appropriate dosage depends on the subject's health, the repairof the lytic bone lesion and prevention of tumor progression, the routeof administration and the formulation used.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

EXAMPLE 1 Patients

174 patients with newly diagnosed multiple myeloma, 16 patients withmonoclonal gammopathy of undetermined significance, 9 with Waldenström'smacroglobulinemia, and 45 normal persons were studied. Table 1 shows thecharacteristics of the patients with multiple myeloma.

TABLE 1 Myeloma patient characteristics and their relationship to MRIlesions Variable n/N % MRI = 1+ MRI = 0 P value Age ≧65 yr 23/169 1417/132 (12.9%)  6/36 (16.7%) 0.59* Caucasian 147/169  87 113/132(85.6%)  33/36 (91.7%) 0.42* Female 68/169 40 55/132 (41.7%) 13/36(36.1%) 0.55 Kappa light chain 104/165  63 79/128 (61.7%) 24/36 (66.7%)0.59 Lambda light chain 61/165 37 49/128 (38.3%) 12/36 (33.3%) 0.59 IgAsubtype 39/169 23 25/132 (18.9%) 14/36 (38.9%) 0.012 B2M ≧4 mg/L 60/16936 47/132 (35.6%) 13/36 (36.1%) 0.96 CRP ≧4 mg/L 12/166 7 11/129 (8.5%) 1/36 (2.8%) 0.47* Creatinine ≧2 mg/dL 19/169 11 16/132 (12.1%) 3/36(8.3%) 0.77* LDH ≧190 UI/L 52/169 31 44/132 (33.3%)  8/36 (22.2%) 0.20Albumin <3.5 g/dL 23/169 14 19/132 (14.4%)  4/36 (11.1%) 0.79* Hgb <10g/dL 40/169 24 31/132 (23.5%)  8/36 (22.2%) 0.87 PCLI ≧1% 23/150 1518/119 (15.1%)  4/30 (13.3%) 1.00* ASPC ≧33% 109/166  66 82/129 (63.6%)26/36 (72.2%) 0.33 BMPC ≧33% 104/166  63 79/129 (61.2%) 24/36 (66.7%)0.55 Cytogenetic 52/156 33 45/121 (37.2%)  6/34 (17.6%) 0.032abnormalities CA13 or 33/52  63 31/121 (25.6%) 3/34 (8.8%) 0.037hypodiploid Other CA 19/52  37 53/103 (51.5%) 16/32 (50.0%) 0.89 FISH1369/136 51 103/136 (75.7%)  28/36 (77.8%) 0.80 Osteopenia 131/173  76 1+Lesions by MRI 137/173  79 3+ Lesions by MRI 108/173  62 1+ Lesions105/174  60 by X-ray 3+ Lesions by X- 69/174 40 ray *Fisher's Exacttest, otherwise Chi-square test

EXAMPLE 2 Bone Imaging

Images were reviewed, without prior knowledge of gene expression data,using a Canon PACS (Picture Archiving and Cataloging System). MRI scanswere performed on 1.5 Tesla GE Signa™ scanners. X-rays were digitizedfrom film in accordance with American College of Radiology standards.MRI scans and x-rays were linked to the Canon PACS system using theACR's DICOM (Digital Imaging and Communications in Medicine) standard.Imaging was done in accordance with manufacturers' specifications. MRIimages were created with pre- and post-gadolinium T1-weighting and STIR(short-tau inversion recovery) weighting.

EXAMPLE 3 Plasma Cell Isolation and Gene Expression Profiling

Following Ficoll-Hypaque gradient centrifugation, plasma cells obtainedfrom the bone marrow were isolated from the mononuclear cell fraction byimmunomagnetic bead selection using a monoclonal mouse anti-human CD138antibody (Miltenyi-Biotec, Auburn, Calif.). More than 90 percent of thecells used for gene expression profiling were plasma cells, as shown bytwo-color flow cytometry using CD138⁺/CD45⁻ and CD38⁺/CD45⁻ markers, thepresence of cytoplasmic immunoglobulin light chains byimmunocytochemistry, and morphology by Wright-Giemsa staining. Total RNAwas isolated with RNeasy Mini Kit (Qiagen, Valencia, Calif.).Preparation of labeled cRNA and hybridization to U95Av2 microarrayscontaining approximately 10,000 genes (Affymetrix, Santa Clara, Calif.)was performed as previously described (Zhan et al., 2002; Zhan et al.,2003). RNA amplification was not required.

EXAMPLE 4 Immunohistochemistry

An antibody from a goat that was immunized against the entire human DKK1protein (R&D Systems, Minneapolis, Minn.) was diluted 1:200 inTris-buffer and added to formalin-fixed, paraffin-embedded bone marrowbiopsy sections for 2 hours at room temperature. Adjacent sections werestained with H & E. Antigen-antibody reactions were developed with DAB(after biotinylated anti-goat antibody [Vector Laboratories, Burlingame,Calif.] [1:400 dilution] and streptavidin-horse radish peroxidase [Dako]staining), and counterstained with Hematoxylin-2.

EXAMPLE 5 Enzyme Linked Immunosorbent Assay (ELISA)

Nunc-Immuno MaxiSorp surface microtiter plates were coated with 50 ml ofanti-DKK1 antibody at 1 mg/ml in 1× phosphate buffered saline, pH 7.2 at4° C. overnight, and blocked with 4 percent bovine serum albumin. Bonemarrow plasma was diluted 1:50 in dilution buffer (1× phosphate bufferedsaline+0.1 Tween-20+1 percent bovine serum albumin). A total of 50 μlwas loaded per well and incubated overnight at 4° C., washed andincubated with biotinylated goat anti-human DKK1 IgG (R&D Systems)diluted to 0.2 mg/ml in dilution buffer, followed by addition of 50 μlof 1:10,000 dilution of streptavidin-horse radish peroxidase (VectorLaboratories), all according to manufacturer's recommendations. Colordevelopment was achieved with the OPD substrate system (Dako) based onmanufacturer's instructions. Serial dilutions of recombinant human DKK1(R&D Systems) were used to establish a standard curve. The cell lineT293, which does not express endogenous DKK1 and T293 with stablytransfected DKK1 (Fedi, et al., 1999) were used to validate the ELISAassay.

EXAMPLE 6 Osteoblast Differentiation Assays

C2C12 mesenchymal precursor cells (American Type Tissue Culture, Reston,Va.) were cultured in DMEM (Invitrogen, Carlsbad, Calif.) supplementedwith 10 percent heat-inactivated fetal calf serum. Alkaline phosphataseactivity in C2C12 cells was measured as described (Gallea, et al., 2001;Spinella-Jaegle, et al., 2001). Cell lysates were analyzed for proteincontent using the micro-BCA assay kit (Pierce, Rockford, Ill.).

EXAMPLE 7 Statistical Analyses

Bone disease in multiple myeloma patients was modeled using logisticregression. Independent variables considered were gene expressionintensity values (average difference calls) from ˜10,000 genes (12,625probe sets) measured using version 5.01 MAS (Affymetrix, Santa Clara,Calif.) from 174 cases of newly diagnosed multiple myeloma. The“Signal”, a quantitative measure of gene expression, for each probe setwas transformed to log₂ before entry into the logistic regression modeland permutation-adjustment analysis. There was no prior hypothesis withregard to genes that might be associated with bone disease in myeloma.As a result a univariate model of bone disease for each of the 12,625probe sets was used. Candidate genes were refined using t-tests withpermutation-adjusted significance levels (Westfall and Young, 1993). TheWestfall and Young analysis was used to adjust for the multipleunivariate hypothesis tests. Group differences in DKK1 signal and DKK1protein levels were tested using the Wilcoxon rank sum test. Significantdifferences in patient characteristics by status of bone disease weretested using either the Fisher's exact test or the chi-square test.Expression intensities of genes identified by logistic regression werevisualized with Clusterview (Golub, et. al., 1999). Spearman'scorrelation coefficient was used to measure correlation of geneexpression and protein levels. Significant differences, in osteoblastdifferentiation, between the control and each experimental conditionwere tested using the Wilcoxon rank sum test; separate comparisons weremade for each unique C2C12 experiment. Two-sided p-values less than 0.05were considered significant and two-sided p-values less than 0.10 wereconsidered marginally significant.

EXAMPLE 8 Gene Expression Profiling of Myeloma Cells

To identify genes that were overexpressed and associated with thepresence of bone lesions, comparing microarray data from patients withor without bone lesions were performed. As MRI-defined focal lesions ofbone can occur before radiologically identifiable lytic lesions,T1-weighted and STIR-weighted imaging to evaluate bone lesions wereused. The gene expression patterns of approximately 10,000 genes inpurified plasma cells from the marrow of patients with no bone lesions(n=36) and those with 1 or more (1+) MRI-defined focal lesions (n=137)were modeled by logistic regression analysis. The model identified 57genes that were expressed differently (P<0.0001) in the two groups ofpatients (FIG. 1A). These 57 genes were further analyzed by t-tests withpermutation-adjusted significance (Westfall and Young, 1993). Thesestatistical tests showed that 4 of the 57 genes were overexpressed inpatients with 1+MRI lesions: dihydrofolate reductase (DHFR), proteasomeactivator subunit (PSME2), CDC28 protein kinase 2 (CKS2), and dickkopfhomolog 1 (DKK1). Given that the gene for the Wnt/β-catenin signalingantagonist DKK1 is the only one of the four that codes for a secretedfactor and that Wnt/β-catenin signaling is implicated in bone biology,further tests on DKK1 were carried out. An analysis of the results fromthe 173 patients with myeloma showed that DKK1 signal for patients with1+MRI and no x-ray lesions differ significantly compared to patientswith no MRI and no x-ray lesions (median signal: 2,220 vs. 285; p<0.001)but does not differ significantly compared to patients with 1+MRI and1+x-ray (median signal: 2,220 vs. 1,865; p=0.63) (FIG. 1B, Table 2).

Monoclonal gammopathy of undetermined significance (MGUS) is a plasmacell dyscrasia without lytic bone lesions and can precede multiplemyeloma. In 15 of 16 cases of MGUS, DKK1 was expressed by bone marrowplasma cells at levels comparable to those in multiple myeloma with noMRI or x-ray lesions of bone (FIG. 1B). DKK1 was undetectable in plasmacells from 45 normal donors, and 9 patients with Waldenström'smacroglobulinemia a plasma cell malignancy of the bone lacking bonelesions (FIG. 1B).

TABLE 2 DKK1 mRNA and protein levels in MRI/X-ray-lesion definedsubgroups of MM No MRI/No X- 1+ MRI/No X- 1+ MRI/1+ X- ray ray ray N 3633 104 DKK1 (Signal) Mean (Std) 536.1 (720.7) 3146.5 (3079.9) 3415.1(4870.8) (mRNA) DKK1 (Signal) Min, Median, 19.2, 284.9, 16.4, 2220.2,9.4, 1864.7, (protein) Max 3810.2 10828.4 28859.1 N 18 9 41 DKK1 (ng/ml)Mean (Std) 9.0 (4.7) 24.0 (17.7) 34.3 (75.3) (mRNA) DKK1 (ng/ml) Min,Median, 1.8, 8.7, 19.7 7.4, 20.4, 61.8 2.5, 13.5, 475.8 (protein) Max

EXAMPLE 9 Global Gene Expression Reveals DKK-1 and FRZB Linked to LyticBone Lesion in Multiple Myeloma

In order to further identify the molecular determinants of lytic bonedisease, the expression profiles of ˜12,000 genes in CD138-enrichedplasma cells from newly diagnosed multiple myeloma patients exhibitingno radiological evidence of lytic lesions on bone surveys (n=28) werecompared to those with ≧3 lytic lesions (n=47). The Chi-square test ofabsolute calls (a qualitative measure of gene expression) was used toidentify 30 genes that distinguished the two forms of disease (P<0.05).The Wilcoxon Rank Sum (WRS) test of the signal call (a quantitativemeasure of gene expression) revealed that 104 genes (49 up- and 55down-regulated) differentiated the two disease subtypes (P<0.001).

The Chi-square test identified the RHAMM proto-oncogene as the mostsignificant discriminator between the two groups. It was expressed inonly 7 of 28 patients with no bone disease compared with 34 of 47patients with bone disease (FIG. 2). As expected, plasma cells from only1 of 11 monoclonal gammopathy of undetermined significance expressedRHAMM (FIG. 3). WRS ranked RHAMM as the 14^(th) most significantdiscriminator between the lytic lesion group and no lytic lesion group.NCALD, a calcium binding protein involved in neuronal signaltransduction, was present in 11/28 (40%) of no lytic lesion group butonly in 2/47 (4%) lytic lesion group. Other notable genes identified byChi-square analysis included FRZB, an antagonist of Wnt signaling, thatwas present in 40/47 (85%) of lytic lesion group and 15/28 (53%) of nolytic lesion group. CBFA2/AML1B has been linked to MIP1α expression andwas present in 50% of the no lytic lesion group and in 79% of the lyticlesion group.

PTTG1 (securin) involved in chromosome segregation was identified by WRSas the most significant discriminating gene (P=4×10⁻⁶). It was calledpresent in 11% of no lytic lesion group but present in 50% of the lyticlesion group (FIG. 4). Other notable genes in the WRS test included theTSC-22 homologue DSIPI which was expressed at lower levels in lyticlesion group (P=3×10⁻⁶). DSIPI is also down-regulated in 12 of 12multiple myeloma plasma cells after ex-vivo co-culture with osteoclasts.

In addition, 4 so called “spike genes” were identified that were morefrequently found in lytic lesion group versus no lytic lesion group(p<0.05): IL6, showing spikes in 0/28 no lytic lesion group and 7/47lytic lesion group (p=0.032); Osteonidogen (NID2) showing spikes in 0/28no lytic lesion group and 7/47 lytic lesion group (p=0.032); Regulatorof G protein signaling (RGS13) showing spikes in 1/28 no lytic lesiongroup and 11/47 lytic lesion group (p=0.023); and pyromidinergicreceptor P2Y (P2RY6) showing spikes in 1/28 no lytic lesion group and1/47 lytic lesion group (p=0.023).

Thus, these data suggest that gene expression patterns may be linked tobone disease. In addition to being potentially useful as predictors ofthe emergence of lytic bone disease and conversion from monoclonalgammopathy of undetermined significance to overt multiple myeloma, theymay also identify targets for potential intervention.

EXAMPLE 10 DDK1 and FRZB Tend to be Expressed at Higher Levels in PlasmaCells from Focal Lesions than from Random Marrow

Given the relationship of DKK-1 and FRZB to lytic lesions, DKK-1 andFRZB expressions were compared in plasma cells derived from random bonemarrow aspirates of the iliac crest with those derived by CT-guided fineneedle aspiration of focal lesions of the spine. These results showedsignificantly higher levels of expression in plasma cells from focallesions.

EXAMPLE 11 DKK-1 and FRZB are not Expressed in Plasma Cells fromWaldenstrom's Macroglobulinemia

Waldenstrom's macroglobulinemia is a rare plasma cell dyscrasiacharacterized by a monoclonal IgM paraproteinemia and lymphoplasmacyticinfiltration of bone marrow, lymph nodes and spleen. Its clinicalpresentation is variable as is the clinical course, yet unlike multiplemyeloma, bone lesions are rare. Although global gene expressionprofiling of CD138-enriched bone marrow plasma cells from 10 cases ofWaldenstrom's Macroglobulinemia reveled gross abnormalities, thesecells, like normal bone marrow plasma cells, lack expression of FRZB andDKK (FIG. 20).

EXAMPLE 12 FRZB and Endothelin Receptor B are Correlated with DKK-1

Endothelin 1 is a 21 amino acids vasoconstrictor. Two receptors forendothelin, receptors A and B, have been identified. Breast and prostatecancer cells can produce endothelin 1, and increased concentrations ofendothelin 1 and endothelin receptor A have been found in advancedprostate cancer with bone metastases. Breast cancer cells that producedendothelin 1 caused osteoblastic metastases in female mice. Conditionedmedia and exogenous endothelin 1 stimulated osteoblasts proliferationand new bone formation in mouse calvariae cultures (FIG. 31). Theseresults suggest that endothelin is linked to bone formation.

Table 3 shows that the expression of endothelin receptor B (ENDRB) wascorrelated with that of DKK-1. Endothelin receptor B was a ‘spike’ genein one third of newly diagnosed multiple myeloma (FIG. 29). Endothelinreceptor B was also expressed in subsets of monoclonal gammopathy ofundetermined significance (MGUS) and smoldering multiple myeloma but notin normal plasma cells (FIG. 30).

TABLE 3 Correlation Between Endothelin Receptor B (EDNRB) and DKK-1 GeneSymbol Asymp. Significance (two-tailed) DKK-1 6.35 × 10⁻¹⁴ FRZB 6.59 ×10⁻⁸ EDNRB 0.00014 DKFZP564G202 4.83 × 10⁻¹¹ IFI27 1.43 × 10⁻⁶ SLC13A30.00011 CCND1 0.00010 SYN47 4.27 × 10⁻¹⁰ PCDH9 0.00029

EXAMPLE 13 In Vivo Drug Treatment Upregulates DKK-1

DKK-1 expression is massively upregulated by UV irradiation and severalother gentoxic stimuli. To see if multiple myeloma plasma cells alsoupregulate the genes in response to drugs used to treat this disease,gene expression profiling of multiple myeloma plasma cells was performedbefore and after 48 hour in vivo treatment with thalidomide (FIG. 33),ImiD (FIG. 34), PS-341 (FIG. 32), or dexamethasone (FIG. 35). These datashowed that DKK-1 and FRZB expression could be massively upregulated inmany cases and thus supporting a direct role of DKK-1 in triggeringapoptosis of multiple myeloma plasma cells. It is interesting to notethat a newly diagnosed patient who was primary refractory to all agentstested showed low levels of DKK-1 in initial prestudy tests and nevershowed increased expression of DKK-1 or FRZB after drug treatment,supporting a role for DKK-1 expression in promoting apoptosis ofmultiple myeloma plasma cells. In support of this notion, DKK-1 and FRZBwere expressed at low to undetectable levels in 30 HMCL and severalcases of extramedullary disease (FIG. 15).

EXAMPLE 14 Co-Culture of Multiple Myeloma with Osteoclasts Results inMassive Downregulation of JUN, FOS, and DKK-1

The close relationship between myeloma cells and osteoclasts isexpressed clinically by the association of debilitating lytic bonedestruction with multiple myeloma. The development of lytic bone lesionsis caused by the activation of osteoclasts through direct and indirectinteractions with myeloma plasma cells. The critical role of osteoclastsin the survival and growth of myeloma cells and in sustaining thedisease process has been gleaned clinically and demonstrated in vivo inexperimental models such as the SCID-hu model for primary human myeloma.

In order to investigate the molecular consequences of multiple myelomaplasma cell/osteoclast interactions, an ex vivo system was developed inwhich CD138-enriched multiple myeloma plasma cells were co-cultured withosteoclasts derived from multiple myeloma peripheral blood stem cells orPBSCs and MNC from healthy donors. CD138-enriched multiple myelomaplasma cells co-cultured with human osteoclasts derived from peripheralblood stem cells from normal donors or multiple myeloma patientsmaintained their viability and proliferative activity as indicated byannexin V flow cytometry, BrdU labeling index and [³H]TdR incorporationfor as long as 50 days. Purity level of plasma cells before and afterco-cultures was greater than 95% as determined by CD38/CD45 flowcytometry.

Microarray analyses of the expression of ˜12,000 genes in 12 multiplemyeloma plasma cells were performed before and after 4 day co-culture.Heirarchical cluster analysis of the 12 multiple myeloma plasma cellspairs and 150 newly diagnosed multiple myeloma plasma cells using 7,913probes sets (genes) revealed that whereas the pre-co-culture sampleswere distributed amongst 3 major cluster groups, the post-co-culturesamples clustered tightly together in 2 of the major branches. Ananalysis of the significant gene expression changes after co-cultureshowed that 95 probe sets (genes) changed 2- to 50-fold (77 up- and 18down-regulated) in at least 8 of the 12 multiple myeloma plasma cellsafter co-culture. CD138-enriched plasma cells from 5 healthy donorsshowed identical shifts in many of the same genes, suggesting thatmultiple myeloma plasma cells do not exhibit altered responses toosteoclasts. However, normal plasma cells as opposed to their malignantcounterparts did not survive in long term co-cultures with osteoclasts.

The most striking changes were in the up-regulation of the chemokinesGRO1, GRO2, GRO3, SCYA2, SCYA8, SCYA18, and IL8. Other notable genesincluded the chemokine receptor CCR1, osteopontin (SPP1), the integrinsITGB2 and ITGB5, matrix metalloproteinase 9 (MMP9), cathepsin K (CTSK)and cathepsin L (CTSL). Surprisingly, a large number ofosteoclast-related genes were among the 77 up-regulated genes. Thedown-regulated genes included cyclin B (CCNB1), the cyclin B specificubiquitin ligase UBE2C, the TSC-22 homologue DSIPI, and JUN, JUND, FOS,and FOSB.

Gene expression changes were also tested in 10 osteoclast cultured aloneand after co-culture with multiple myeloma plasma cells. Twenty-fourgenes (14 up- and 10 down-regulated) changed 2- to 10-fold in at least 7of 10 osteoclasts after co-culture. There were no significantdifferences in gene expression between multiple myeloma plasma cellscultured with osteoclasts derived from multiple myeloma patients or fromhealthy donors, suggesting that multiple myeloma osteoclasts are notqualitatively different than those derived from normal donors.

No significant changes in gene expression were observed when multiplemyeloma plasma cells were cultured in media derived from a co-cultureexperiment, suggesting that contact is important. Given the low ratio ofmultiple myeloma plasma cells to osteoclasts in the co-cultureexperiments (1000:1), it is unlikely that all plasma cells can be incontact with the osteoclasts simultaneously. Thus, it is likely thatsome intercellular communication between multiple myeloma plasma cellsin contact with osteoclasts and those other multiple myeloma plasmacells occurs.

It is known that osteoclasts play a major role in multiple myeloma bonedisease as well as providing multiple myeloma with anti-apoptoticsignals. Recent studies have shown that JUN directly regulates DKK-1expression and that JUN and DKK-1 control apoptosis.

To determine if osteoclasts may prevent apoptosis of multiple myelomaplasma cells by modulating JUN and DKK-1, gene expression profiling wasperformed on purified plasma cells from 12 primary multiple myelomacases before and after 48 hours of co-culture with in vitro derivedosteoclasts. Multiple myeloma plasma cells in the co-culture hadsignificantly higher long-term viability than cells cultured alone. Geneexpression profiling of multiple myeloma plasma cells before and afterosteoclast co-culture revealed that JUN, FOS, and FOSB were 3 of 40genes down-regulated more than 2-fold in all cases (n=12/12).Hierarchical cluster analysis of HMCL and primary multiple myeloma cellswith 95 genes significantly modulated in multiple myeloma plasma cellsafter co-culture revealed a striking similarity between HMCL, primarymultiple myeloma co-cultured with osteoclasts and a subset of newlydiagnosed multiple myeloma in that these cell types had relatively lowlevels of c-JUN and c-FOS.

Importantly, whereas primary multiple myeloma cells show a high degreeof spontaneous apoptosis when cultured alone, multiple myeloma plasmacells cultured in the presence of osteoclasts can survive indefinitely.These data support a link between JUN and DKK-1 and also suggest thatloss of JUN and DKK expression in multiple myeloma may be associatedwith disease progression as extramedulalary disease and HMCL, which areinvariably derived from extramedullary disease, lack both JUN and DKK.It is interesting to speculate that one of the major influences ofosteoclasts on multiple myeloma growth and behavior is to downregulateJUN and DKK-1, which directly affects plasma cells apoptosis. Treatmentof HMCL and primary multiple myeloma/osteoclasts co-cultures with DKK-1is expected to result in apoptosis of multiple myeloma plasma cells.DKK-1 will likely have no effect on the osteoclasts, as these cells donot express the Wnt co-receptor LRP-5. Normal bone marrow derived plasmacells also do not express DKK-1 and may help explain their long-livednature.

EXAMPLE 15 Synthesis of DKK1 Protein by Plasma Cells

Serial sections from bone marrow biopsies of 65 cases of multiplemyeloma were stained for the presence of DKK1. The plasma cells in thesecases contained DKK1 in a manner consistent with the gene expressiondata (FIG. 39). Similar experiments with biopsies from 5 normal donorsfailed to identify DKK1 in any cell. There was a strong tendency forDKK1 positive myelomas to have low-grade morphology (abundant cytoplasmwithout apparent nucleoli) with an interstitial growth pattern. Thisstaining was found to be greatest in plasma cells adjacent to bone. DKK1negative myelomas tend to bear high-grade morphology (enlarged nucleiand prominent nucleoli) with a nodular or obliterative growth pattern.In biopsies with an interstitial growth pattern, DKK1 was either present(in varying percentages of cells) or absent. In contrast, myelomas withthe more aggressive nodular growth patterns DKK1 was uniformly absent.Importantly, in cases with both interstitial and nodular growth, theinterstitial cells were positive and the nodular cells negative.

EXAMPLE 16 DKK1 Protein in Bone Marrow Plasma

An enzyme-linked immunosorbent assay (ELISA) showed that theconcentration of DKK1 protein in the bone marrow plasma from 107 of the173 newly diagnosed multiple myeloma patients for which gene expressiondata was also available, was 24.02 ng/ml (S.D. 49.58). In contrast, DKK1was 8.9 ng/ml (S.D. 4.2) in 14 normal healthy donors, 7.5 ng/ml (S.D.4.5) in 14 cases of MGUS, and 5.5 ng/ml (S.D. 2.4) in 9 cases ofWaldenström's macroglobulinemia. DKK1 gene expression and the level ofDKK1 in the bone marrow plasma were positively correlated (r=0.65,P<0.001) in the 107 cases of myeloma (FIG. 40A). There was also a strongcorrelation between DKK1 protein levels in bone marrow plasma andperipheral blood plasma in 41 cases of myeloma in which both sampleswere taken simultaneously (r=0.57, P<0.001).

In 68 patients in whom both DKK1 protein levels in the bone marrowplasma and the presence of bone lesions were determined, DKK1 protein inpatients with 1+MRI and no x-ray lesions differ significantly comparedto patients with no MRI and no x-ray lesions (median level: 20 ng/ml vs.9 ng/ml; p=0.002), but does not differ significantly compared topatients with 1+MRI and 1+x-ray lesions (median level: 20 ng/ml vs. 14ng/ml; p=0.36) (FIG. 40B, Table 2).

EXAMPLE 17 Effect of Bone Marrow Serum on Osteoblast Differentiation InVitro

Bone morphogenic protein-2 can induce differentiation of the uncommittedmesenchymal progenitor cell line C2C12 (Katagiri, et al., 1994) intoosteoblasts through a mechanism that involves Wnt/b-catenin signaling(Bain, et al., 2003; Roman-Roman, et al., 2002). Alkaline phosphatase, aspecific marker of osteoblast differentiation, was undetectable in C2C12cells grown in 5 percent fetal calf serum for 5 days (FIG. 41A).Treatment of C2C12 cells with 50 ng/ml of BMP-2 for 5 days induced themto produce alkaline phosphatase, whereas alkaline phosphatase was notproduced by C2C12 cells that were concomitantly cultured with BMP-2 and50 ng/ml recombinant human DKK1. This in vitro effect on alkalinephosphatase production was neutralized by a polyclonal anti-DKK1antibody, but not by a non-specific polyclonal goat IgG. Bone marrowserum with a DKK1 concentration >12 ng/ml from five patients withmyeloma inhibited the production of alkaline phosphatase by C2C12 cellstreated with BMP-2, and this effect was reversed by the anti-DKK1antibody, but not by non-specific IgG (FIG. 41B). By contrast, C2C12cells treated with 50 ng/ml BMP-2 and 10 percent serum from the bonemarrow of a normal donor induced the production of alkaline phosphataseby the cells (FIG. 41B).

EXAMPLE 18 The Functional Role of Canonical Wnt Signaling and DKK1Inhibition of this Pathway in Bone Morphogenic Protein (BMP)-2-InducedOsteoblast Differentiation

Expression of the DKK1 by multiple myeloma cells has been shown tocorrelate with lytic bone disease in multiple myeloma. Furthermore, asdiscussed supra, it was observed that alkaline phosphatase productionwas inhibited in presence of recombinant human DKK1. Hence, the presentinvention further investigated the mechanism by which DKK1 contributedto this process.

Cells and Cell Culture:

Mouse pluripotent mesenchymal precursor cell line C2C12 and the humanosteoblast cell line hFOB1.19 were purchased from America Type CultureCollection (Manassas, Va.). C2C12, MG63, Saos-2, and 293T cells werecultured in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen,Carlsbad, Calif.) containing 10% heat-inactivated FBS, penicillin (100U/ml streptomycin (100 mg/ml), and 4 mM L-glutamine. Cells weremaintained at 37° C. and humidified with 95% air and 5% CO₂ for cellculture. hFOB1.19 was cultured in a 1:1 mixture of Ham's F12 and DMEMwith 10% FBS in the presence of 0.3 mg/ml G418.

Constructs and Transfectants:

To generate dominant negative (DN)-b-catenin stable clones, C2C12 cellswere transfected with pcDNA4 vector or a vector containingDN-beta-catenin cDNA (Boyden et al., 2002) using lipofectimine(Invitrogen) following manufacturer's instructions. After transfection,stable clones were generated by growing the cells in DMEM containing 10%FBS in the presence of Neocin (1 mg/ml) for two weeks. Stable Dkk1 andDkk2 expressing clones generated in C2C12 and OPM-2 cells werepreviously described (Qiang et al., 2003).

Preparation of Conditioned Medium:

Conditioned medium (CM) containing Wnt3a, Dkk1, Dkk2 and or appropriatecontrol constructs was prepared as previously described (Qiang et al.,2003). Dkk1 and Dkk2 proteins in CM were detected by immunoblottingusing anti-V5 (explain what anti-V5 is specific for)) and anti-Dkk1antibodies by ELISA. The supernatant from culture medium wasconcentrated five-fold by using a YM-30 column (Qiang et al., 2002).Dkk1 levels in bone marrow plasma from MM patients was detected byELISA.

Immunoblotting Analysis:

Cells were incubated in MEM, Wnt3a CM, control CM, or with recombinantWnt3a for indicated times. For inhibition studies, cells were pretreatedwith purified recombinant Dkk1 at indicated concentrations or Dkk1 CM,Dkk2 CM, or bone marrow plasma with low or high concentrations of Dkk1protein for one hour. Following treatment, cells were lysed as described(Qiang et al., 2002). Cell lysates were separated by SDS-PAGE andtransferred to Immobilon polyvinylidene difluoride membranes (Millipore,Bedford, Mass.). Immunoblotting was performed using the indicatedantibodies.

GST-E-Cadherin Binding Assay:

The GST-E-cadherin binding assay was performed as described (Bafico etal., 1998). Briefly, the beta-catenin binding site of E-cadherin as aGST-fusion protein was purified using GST beads. GST-E-cadherin was usedto precipitate uncomplexed b-catenin present in 500 mg of cell lysate.Precipitated b-catenin was detected by immunoblotting using a b-cateninmonoclonal antibody. Non-phosphorylated b-catenin was detected with amonoclonal antibody specific for b-catenin dephosphorylated at residuesof 27-37 (Alexis, San Diego, Calif.).

Enzyme-Linked Immunosorbent Assay:

Microtiter plates were coated with 50 μl of anti-Dkk1 antibody (R&DSystems, Minneapolis, Minn.) according to manufacturer recommendations.Bone marrow plasma (1:50) in dilution buffer was added and incubatedovernight at 4° C. Plates were washed and incubated with biotinylatedgoat anti-human Dkk1 IgG (R&D Systems, Minneapolis, Minn.) followed bystreptavidin-horseradish peroxidase (Vector Laboratories), according tomanufacturer recommendations.

Luciferase Reporter Gene Assay:

Cells plated at 5×10⁴ per well in a 12-well plate were transientlyco-transfected with 1 mg/ml of either TOPflash, FOPflash (Korinek etal., 1997), or Cbfa-1-luc (kindly provided by Dr. Ying Zhang, NCI, NIH)and 50 ng of pSV-b-galactosidase vector to normalize for transfectionefficiency using Lipofectamine according to manufacturer instructions(Invitrogen). Following transfection, cells were exposed to Wnt3a CM orcontrol CM for 24 hr prior to luciferase assay. Luciferase activity wasmeasured as previously described (Qiang et al., 2003).

Alkaline Phosphatase (ALP) Assay:

Cells were cultured in DMEM with 2% horse serum including either BMP-2(200 ng/ml), Wnt3a CM, BPM-2 plus Dkk1, or BMP-2 plus Wnt3a CM for 72 or96 hr followed by lysis in 150 ml of lysis buffer (20 mM Tris HCl, pH 8and 150 mM NaCl, 0.2% NP40). ALP activity was measured using ALP kit(Diagnostic Chemical Limited, Exton, Pa.) according to manufacturerinstructions. Absorbance The of samples was determined with a SpectraMax340 Microplate Spectrophotometer (Molecular Devices, Sunnyvale,Calif.) at 402 nm. Cell lysates were analyzed for protein content withusing the micro-BCA assay kit (Pierce, Rockford, Ill.).

RT-PCR Analysis:

First strand cDNA synthesis was performed as previously described. (31)All PCR reactions began with a first cycle at 95° C. for 3 min and afinal cycle at 72° C. for 10 minutes with an additional 35 cycles at 94°C./30 s, 60° C./45 s, 72° C./1 min. Primer sequences for the indicatedhuman genes are as described (Qiang et al., 2003). Primers, including Fz(Table 4), TCF and Dkk (Table 5) were designed using a primer pairprogram in the MacVector (City) software based on gene sequences fromthe NIH Gene Bank (www.ncbi.nlm.nih.gov).

TABLE 4 Mouse Frizzled Oligonucleotide Primers for RT-PCR NucleotidePrimer Orientation Nucleotide Seq 5′ to 3′ Position SEQ ID NO Fz1F Senseatgtgtatgtgcgtgtggaccg 2530-2551 1 Fz1R Anti-sensegggagatgctgaaggaaatgacc 2854-2832 2 Fz2F Sense aaataggttgggttggagggag3077-3098 3 Fz2R Anti-sense aaacaggagagacggttgagagcg 3537-3514 4 Fz3FSense tattgaggaggatggaaccagtgc 2286-2309 5 Fz3R Anit-sensecaaagcagtcaccacacatagagg 2607-2584 6 Fz4F Sensetagttggatgccgatgaactgactg 1394-1417 7 Fz4R Anti-sensettccccctcttctctctctttacc 1865-1842 8 Fz5F Sense acattcgccaccttctggattg1568-1589 9 Fz5R Anti-sense ttttggttgcccacatagcag 2058-2038 10 Fz6FSense aatggacacttttggcatccg 635-655 11 Fz6R Anti-sensectctgggtatctgaatcgtctaacg 1001-977 12 Fz7F Sense aagggggaaactgcggtatg1843-1862 13 Fz7R Anti-sense tctctctctctgctggtctcaacc 2181-2158 14 Fz8FSense tccatctggtgggtaatcctgtc 1225-1247 15 Fz8R Anti-sensecggttgtgctgctcatagaaaag 1667-1645 16 Fz9F Sense cgcccgattatcttcctttctatg998-1021 17 Fz9R Anti-sense tagcagagcccagtcagttcatc 1371-1349 18 Fz10FSense ccaacaagaacgaccccaactac 528-550 19 Fz10R Anti-senseaagaagcacagcacggaccagatg 834-811 20

TABLE 5 Mouse TCF and Dkk oligonucleotide Primers for RT-PCR NucleotidePrimer Orientation Nucleotide Seq 5′ to 3′ Position SEQ ID NO TCF1FSense acgaacatttcagcagtccacac 197-219 21 TCF1R Anit-sensegcattgaggggtttcttgatgac 629-607 22 TCF3F Sense caacgaatcggagaatcagagc208-229 23 TCF3R Anti-sense atggcgaccttgtgtccttgac 509-488 24 TCF4FSense tgcctggtgggtgaaaaatgc  96-115 25 TCF4R Anti-sensecttgagggtttgtctgctctgg 561-540 26 LEF1F Sense ttctctttttctcccctccccc288-309 27 LEF1R Anit-sense aaacctctccacggattcctcg 567-546 28 Dkk1FSense acattcgccaccttctggattg 1501-1525 9 Dkk1R Anti-sensegcaaaagcaccaaccacacttg 1797-1776 29 Dkk2F Sense aatgcggaagaatgagggatg1593-1613 30 Dkk2R Anti-sense tgccaatctgaaggaaatgcc 1839-1819 31 Dkk3FSense cgtggacttggcaaaatgtaacc 1511-1533 32 Dkk3R Anti-sensegagcactggctttcagaggtattg 1937-1914 33 Dkk4F Sense aagccccagaaatcttccagc697-717 34 Dkk4R Anti-sense tgaacacaacaacaagtcccgtg 839-817 35Sub-Cloning of PCR Fragments and DNA Sequence Analysis:

PCR fragments were subcloned using TOPO-TA cloning vector according tomanufacturer instructions (Invitrogen) and sequence analysis performedas previously described (Qiang et al., 2003). Data analysis wasperformed using MacVactor software and comparisons made with NCBI BLAST(http://www.ncbi.nlm.nih.gov/blast/).

Real-Time Quantitative PCR:

One microgram of total RNA was reverse transcribed into total cDNA.Quantitative PCR (qPCR) was performed using an ABI Prism 7000 sequencedetection system (Applied Biosystems, Foster City, Calif.). The reactionmixture contained 1 ml of cDNA, dedicated buffers with specific primersand probes (5′-labeled by 6-carboxy-fluorescein and 3′-labeled by1-carboxy-teteramethyrhdamine), and DNA polymerase in a total 20 mlvolume. Following 2 min incubation at 50° C. and 10 min incubation at95° C. for denaturing, the reaction was subjected to 40-cycleamplification at 95° C. for 15 second to denature and at 60° C. for 1min for annealing/extension. Each cDNA sample was analyzed in triplicatein parallel with GAPDH as a control. Changes in mRNA concentration weredetermined by subtracting the CT (threshold cycle) of target gene fromthe CT of GAPDH (Δ=CT gene−CT GAPDH). The mean of Δ control wassubtracted from the ΔSiLRP5/6 reaction (mean Δ control−ΔSiLRP5/6=e) Thedifference was calculated as 2^(e) by the ^(2−ΔΔC) _(T) (35).

RNA Interference:

Chemical synthesis of siRNA specific to LRP5/6, GFP, and control siRNAwere purchased from Qiagen (Valencia, Calif.). The siRNA weretransiently transfected into C2C12 using Lipofectamine according tomanufacturer instructions (Invitrogen). RNA was isolated after 24, 48 or72 hours then subjected to RT-PCR or qPCR for determination of efficacyof target gene silencing.

Statistical Analysis:

Statistical significance of differences between experimental groups wasanalyzed by a Student's t-test using the Microsoft Excel softwarestatistical package. Significant p values were less than 0.05 bytwo-tailed test.

Expression of Wnt Receptors and Co-Receptors in OB Cells:

RT-PCR was used to evaluate the presence of Wnt receptor mRNA in C2C12,hFO1.19, and two human osteoblast-like cells lines, MG63 and Saos-2.Analysis using primers for all Fz family members (FIGS. 42A-42D)revealed expression of Fz1, 2, 4, 5, 6, 7, 8, and 9 with relativelyhigher levels of Fz1, 6, and 7 in C2C12 cells. Similar expression ofmultiple Fzs was observed in the human lines. Fz3 was expressed in allhuman lines, but not C2C12, while Fz6 was expressed in C2C12 but none ofthe human lines. LRP5 and LRP6 were identified in all mouse and humanlines indicating. Thus, both components of functional Wnt receptors areexpressed in OB cell lines and the presence of multiple receptors islikely.

Canonical Wnt Signaling is Activated in Pre-Osteoblast Cell Lines:

Having demonstrated the presence of Fz and LRP receptors, we sought todetermine whether a functional canonical Wnt/b-catenin pathway waspresent by first examining the status of downstream b-catenin. Becauseosteoblasts express high levels of cadherin proteins (includingb-catenin) (Cheng et al., 1998), especially in the form ofmembrane-bound protein (Nelson and Nusse, 2004), the GST-E-cadherinbinding assay was used to separate cytosolic, free (uncomplexed)beta-catenin from the membrane bound form. Examination of Wnt3atreatment effects revealed significant increases of free b-cateninappearing in a time-dependent manner (FIG. 42E) in all cell lines.Increases in beta-catenin levels were apparent at 8 hr and remainedelevated for 24 hrs. Increases in beta-catenin correlate with thepresence of the non-phosphorylated, transcriptionally active form of theprotein (van Noort et al., 2002) as determined by analysis withantibodies specific to non-phosphorylated b-catenin (FIG. 42F).

RT-PCR analysis of TCF/LEF family members revealed expression of TCF1,3, 4, and LEF1 mRNA in C2C12 cells (FIG. 42G). Similar results ofmultiple TCF/LEF family member expression were observed in the humanlines although LEF1 appeared not to be expressed in MG63 (FIG. 42H).Transient transfection of cells with TOPflash reporter constructscontaining binding sites for the b-catenin/TCF/LEF transcription complexresulted in a significant increase in luciferase activity in thepresence of Wnt3a CM. In contrast, no transcriptional activation wasobserved in control cells treated with L929 CM or cells transfected withFOPflash containing mutant TCF/LEF biding sties (FIG. 42I). Takentogether, these results indicate that a canonical Wnt signaling pathwayis functional in pre-osteoblast cells.

Dkk1 and MM Patient Sera Inhibit Wnt3a Induced Beta-Catenin inPre-Osteoblasts:

To examine the effect of Dkk1 in OBs, cells were incubated withincreasing amounts of Dkk1 prior to Wnt3a treatment. As shown in FIG.3A, pretreatment with Dkk1 led to dose-dependent inhibition ofWnt3a-increased, non-phosphorylated b-catenin in C2C12, hFOB1.19 andSaos-2 cells. Dkk1 inhibited non-phosphorylated b-catenin atconcentrations beginning at 25 ng/ml with maximal inhibition at 50 ng/mlin C2C12. In hFOB1.19 and Saos-2 pronounced inhibition was observed atthe lowest concentration tested (5 ng/ml) which changed only moderatelywith increasing concentration. Similar results were observed in analysisof total free b-catenin (not shown).

To determine whether Dkk1 expression by MM cells might have similareffects on functional Wnt signaling in the bone marrow microenvironment,the following experiments were performed. First, stable Dkk1-expressingclones were generated in the OPM2 mM cell line and lysates containingDkk1 used to determine the effect on Wnt3a-induced stabilization ofb-catenin in C2C12 cells. The presence of Dkk1 protein in cell lysatesfrom stable OPM-2 clones was determined by Western blot analysis withanti-V5 antibody (FIG. 42K). Dkk1 CM from OPM-2/Dkk1-expressing clonesinhibited accumulation of uncomplexed, non-phosphorylated b-catenin inC2C12 cells (FIG. 42L). Furthermore, bone morrow plasma from four MMpatients containing over 100 ng/ml of Dkk1 (designated H1 to H4)similarly inhibited b-catenin (FIG. 42M). These results suggest thatDkk1 produced from MM cells and MM bone marrow can negatively regulateWnt signaling in pre-osteoblasts.

Dkk1 Inhibits BMP-2-Induced ALP Activity:

Given the characterization of a functional Wnt signaling pathway inmouse and human pre- and osteoblast-like cells, experiments wereundertaken to identify the biological effects associated with thispathway. C2C12 cells were selected as a model since following reasons.First, they undergo pre-osteoblast differentiation in the presence ofBMP-2 (Nishimura et al., 1998). Second, they express less Dkk1 mRNA andprotein, compared with other human cell lines and primal humanmesenchymal cells and finally they react with addition of Dkk1 moresensitively that human other lines (FIG. 42J). As expected, BMP-2treatment led to increased ALP activity whereas Wnt3a alone had noeffect (FIG. 42N). C2C12 cells treated with both Wnt3a and BMP-2demonstrated no obvious increase in ALP activity over BMP-2 aloneindicating a lack of synergy between these factors in ALP production.Thus, exogenous Wnt3a alone is not sufficient to induce C2C12differentiation. However, since Wnt3a mRNA (data not shown) and abundantsteady state levels of b-catenin were detected in C2C12 cells (FIGS.42E-42M), the possibility of a cooperative role between BMP and Wntpathways in C2C12 differentiation was next examined using Dkk1 proteinto block canonical Wnt-beta-catenin signaling. Interestingly,pretreatment of C2C12 cells with 100 ng/ml of Dkk1 significantlyinhibited ALP activity in the presence or absence of Wnt3a (FIG. 42N).Comparable studies in the human cell lines hFOB1.19 and Saos-2 cellsproduced similar results (FIGS. 42O and 42P). These findings suggestthat an autocrine b-catenin loop is critical to BMP-2-mediatedpre-osteoblast differentiation.

An Autocrine Wnt Loop is Required for Differentiation of C2C12 Cells:

To address the role of autocrine Wnt-b-catenin signaling inpre-osteoblast differentiation, C2C12 cells were transfected with adominant negative (DN)-b-catenin construct which lacks TCF/LEF bindingsites and inhibits transcriptional activity (Chung et al., 2002).DN-beta-catenin expression was confirmed by western blot analysis withanti-X-press antibody (FIG. 42Q) and DN-beta-catenin clones exhibitedsignificantly reduced Wnt3a mediated luciferase activity (FIG. 42R).Importantly, marked reductions in ALP activity were observed inDN-b-catenin C2C12 cells in the presence of BMP-2 (FIGS. 42S and 42T).These results suggest that canonical beta-catenin signaling contributesto BMP-2-induced differentiation in C2C12 cells. To further confirm theeffect of Dkk1 on osteoblast differentiation, we ectopically expressedDkk1 and Dkk2 in C2C12 cells. Overexpression of both Dkk1 and Dkk2inhibited endogenous non-phosphorylated beta-catenin (FIG. 42U). Yet,exposure of these clones to medium containing 200 ng/ml of BMP-2significantly inhibited the ALP activity in both Dkk1 and Dkk2 clones,compared with cells expressing empty vector (FIG. 42V). These resultssuggest that Dkk1 and Dkk2 inhibit mesenchymal cell differentiation byinhibition of autocrine Wnt-teta-catenin pool in pre-osteoblasts.

Inhibition of ALP Activity by siRNA Specifically Targeting LRP5/6:

To determine the specific role of Wnt receptors in mesenchymal celldifferentiation in vitro, we used siRNA specific for LRP5 and LRP6 todown regulate gene expression. As shown in FIG. 42W, LRP5 and LRP6 mRNAexpression in C2C12 cells was inhibited by the homologous (siLRP5 andsiLRP6), but not by heterogeneous siRNA (control siRNA). Furthermore,inhibition of either LRP5/6 by their respective siLRNA's occurred in adose-dependent manner as determined by qPCR (FIGS. 42X and 42Y) andneither reduced expression of the other. Treatment of siLRP5- andsiLRP6-expressing cells with BMP-2 resulted in significant reductions inALP activity compared with cells expressing control siRNA (FIG. 42Z)indicating that LRP co-receptors are likely required for pre-osteoblastdifferentiation.

Dkk1 Inhibition of Pre-Osteoblast Differentiation is Independent of theSmad/Cbaf1/Runx2 Pathway:

To address the question of whether the Wnt and BMP-2 pathways wereinvolved in cross-regulation and co-regulation of downstream elementsand/or if Dkk1 directly interferes with BMP-2 signaling to inhibitosteoblast differentiation, the effects of BMP-2 on b-cateninstabilization were first analyzed. As shown in FIG. 42AA, treatment ofall pre-osteoblast cell lines with BMP-2 for 8, 24, and 48 hrs did notresult in changes in b-catenin as compared to Wnt3a treated controls.BMP-2 alone did not induce TCT/LEF transcriptional activity, nor did itsynergize Wnt3a-stimulated TCF/LEF transcriptional activity asdetermined by luciferase activity in C2C12 cells transfected withTOPflash constructs (FIG. 42BB). Thus, BMP-2 did not activate theWnt-beta-catenin pathway at the beta-catenin and TCF/LEF levels.Experiments were next implemented to determine whether Wnt and Dkk1directly regulate BMP-2 signaling. First, BMPR-I and -II wereimmunoprecipitated from C2C12 cell lysates followed by blotting withantibodies to LRP5 and LRP6. Complexes of LRP5/6 and BMPR-I/-II were notfound following either Wnt3a or Dkk1 treatment. Similar results wereobtained in 293T cells transiently transfected with plasmid containingLRP5/6 cDNA. To ascertain whether Wnt3a can activate BMP-2 downstreamtargets, Smad phosphorylation was analyzed using antibodies specific top-Smad1-5463/465. Wnt3a did not induce phosphorylation of Smad1 in C2C12cells (FIG. 42BB) nor Smads5 and 8 as assessed with antibodies top-Smad5-5463/463, and p-Smad8-S426/428 in contrast to BMP-2 controls.Because BMP-2 also increases Smad6 gene expression, which serves asinhibitor of BMP-2 signaling (Iton et al., 2001; Wang et al., 2007), thepresent invention examined if Wnt and Dkk1 affects its expression. Asexpected, BMP-2 induced increase in Smad6 mRNA in a time-dependentmanner in C2C12 cells as measured by qPCR analysis (FIG. 42AD). However,neither Wnt3a alone nor Dkk1 affected BMP-2-induced Smad6 geneexpression. Finally, the effect of Wnt3a and Dkk1 on transcriptionalactivity of Cbfa-1/Runx2 was investigated by transiently transfectingC2C12 with a luciferase reporter construct, Cbfa-1-Luc and treating withWnt3a and Dkk1. Cell lysates assayed for luciferase revealed no changein activity indicating the Wnt3 role in osteoblast differentiation isindependent of Cbfa-1/Run2 transcriptional activity. Taken together,these results suggest that Wnt does not activate downstream elements inthe BMP-2 pathway, BMP-2 does not activate downstream elements in theWnt pathway, and Dkk1 does not directly inhibit BMP-2 signaling.

EXAMPLE 19 Effect of Myeloma Derived DKK-1 on Wnt-RegulatedOsteoprotegerin and RANKL Production by Osteoblasts

The present invention examined the influence of DKK1 on RANKL/OPGexpression in myeloma.

Primary Myeloma Cells and Established Myeloma Cell-Lines:

Primary plasma cells (PC) were obtained from heparinized bone marrow(BM) aspirates from multiple myeloma (MM) patients during scheduledclinic visits. Mononuclear cells were isolated from BM of MM patientsusing a Ficoll-Hypaque density gradient centrifugation. FC isolationfrom mononuclear cell fraction was performed by immunomagnetic beadselection with monoclonal mouse antihuman CD138 antibodies using theAutoMACs automated separation system (Miltenyi-Biotec, Auburn, Calif.).PC purity of more than 85% homogeneity was confirmed by 2-color flowcytometry using CD138⁺/CD45 and CD38⁺/CD45 criteria (Becton Dickinson,San Jose, Calif.), immunocytochemistry for cytoplasm light-chainimmunoglobulin (Ig), and morphology by Wright-Giemsa staining.

Cell lines: Human MM cell line, OPM-2 was cultured in RPMI1640 aspreviously described (Qiang et al., 2003). Mouse pluripotent mesenchymalprecursor cell line C2C12 that has the potential of differentiating intoosteoblast in the presence of BMP-2 (Katagiri et al., 1994) and humanosteoblast cell line hFOB1.19 were purchased from America Type CultureCollection (Manassas, Va.). C2C12 and human osteoblast-like cell line,Saos-2 and MG63 were cultured in Dulbecco's Modified Eagle Medium (DMEM)(Invitrogen, Carlsbad, Calif.) containing 10% heat-inactivated FBS,penicillin (100 U/ml), streptomycin (100 mg/ml), and 4 mM L-glutamine.Cells were maintained at 37° C. and humidified with 95% air and 5% CO₂for cell culture.

Coculture System:

C2C12 cells were cultured in 6-well plates in DMEM with 10% FBS andmaintained at subconfluence. MM cells (5×10⁵/ml) were seeded on theC2C12 in the presence or absence of Wnt3a-CM or Cont-CM for indicatedtimes. For coculture with primary cells, CD138 positive cells werecultured on of the C2C12 monolayer for 72 hours in the presence orabsence of rWnt3a with and without anti-DKK1 antibody (R&D System) for48 hours. Total RNA was isolated using TRIZOL reagent (Invitrogen).Supernatants were harvested for protein analysis.

Constructs and Transfectants:

A MM cell line, OPM-2, stably expressing DKK1 was generated aspreviously described (Qiang et al., 2003). Functional DKK1 protein wasdetermined by blocking Wnt3a induced TCF/LEF transcriptional activityusing the TOPflash luciferase assay as previously described. To generatea DKK1 expressing osteoblast cell line, C2C12 cells were transfected,using Lipofectamine (Invitrogen-Life Technologies, Inc.), with a pEF-V5vector or the same vector carrying a DKK1 cDNA, according tomanufacturer's instructions. Clonal cell lines were generated by limiteddilution in growth media containing blasticidin. Positive clones weredetected by anti-V5 antibody with Western blotting analysis. DKK1protein concentration in supernatant cultured positive clones wasmeasured by ELISA analysis. Functional DKK1 protein was determined byanalyzing the effect on stabilization of free beta-catenin as previouslydescribed (Qiang et al., 2007, in press).

Preparation of Conditioned Medium:

Wnt3a conditioned medium (Wnt3a-CM) or control (Cont-CM) was prepared asdescribed (Qiang et al., 2003). Briefly, Wnt3a-producing L cells (stablytransfected with Wnt3a cDNA kindly provided by Dr Shinji Takata) orcontrol L cells were cultured to confluence in DMEM medium supplementedwith 10% FCS after which the medium was replaced with serum-free DMEM.The culture supernatant was collected after 72 hours and designatedWnt3a-CM and Cont-CM, respectively. The concentration of Wnt3a inWnt3a-CM was evaluated by correlating β-catenin stabilization with thatof recombinant Wnt3a (R&D Systems, Minneapolis, Minn.). Theconcentration in 100% CM equates to the 150 to 200 ng/ml of recombinantWnt3a. DKK1 conditioned medium (DKK1-CM) and control medium (Cont-CM)were prepared as described previously. DKK1 protein in DKK1-CM, Cont-CM,supernatant from culture media of C2C12, MG63, and Saos-2 cells, andsera from MM patients were measured by ELISA analysis as describedpreviously.

Immunoblotting Analysis and GST-E-Cadherin Binding Assay:

Proteins from cell lysates derived from C2C12 and OPM-2 cells expressingDKK1 or empty vector were separated by SDS-PAGE and transferred toImmobilon polyvinylidene difluoride membranes (Millipore, Bedford,Mass.). Immunoblotting was performed using the indicated antibodies aspreviously described (Qiang et al., 2005). The GST-E-cadherin bindingassay was performed as described (Bafico et al., 1998). Briefly,proteins were isolated from cells that had been treated with recombinantWnt3a for indicated times. The beta-catenin binding site of E-cadherinas a GST-fusion protein was purified using GST beads. GST-E-cadherin wasused to precipitate uncomplexed beta-catenin in 500 mg of cell lysate.Precipitated beta-catenin was detected by immunoblotting analysis usinga b-catenin monoclonal antibody (Qiang et al., 2005).

Enzyme-Linked Immunosorbent Assay:

Microtiter plates were coated with 50 μl of anti-DKK1 antibody (R&DSystems, Minneapolis, Minn.) according to manufacturer recommendations.Bone marrow serum (1:50) in dilution buffer was added and incubatedovernight at 4° C. Plates were washed and incubated with biotinylatedgoat anti-human DKK1 IgG (R&D Systems, Minneapolis, Minn.) followed bystreptavidin-horseradish peroxidase (Vector Laboratories), according tomanufacturer recommendations. The concentrations of OPG and RANKLproteins in cultured supernatant were measured using the kits fromaccording to manufacturer recommendations (R&D Systems, Minneapolis,Minn.).

RT-PCR Analysis and DNA Sequence Analysis:

Total RNA was isolated using TRIzol reagent (Invitrogen). First strandcDNA synthesis was performed as previously described (Qiang et al.,2003). All PCR reactions began with a first cycle at 95° C. for 3 minand a final cycle at 72° C. for 10 minutes with an additional 35 cyclesat 94° C./30 s, 60° C./45 s, 72° C./1 min. Primers, including human andmouse DKKs were designed using the ‘primer pair program’ using MacVectorsoftware (Qiang et al., 2005) based on gene sequences from the NCBI GeneBank (www.ncbi.nlm.nih.gov). Primer sequences and expected sizes of DNAfragments amplified for the indicated mouse and human genes are listedin Table 6. PCR fragments were subcloned using TOPO-TA cloning vectoraccording to manufacturer instructions (Invitrogen) and sequenceanalysis performed as previously described (Qiang et al., 2003). Dataanalysis was performed using MacVactor software and comparisons madewith NCBI BLAST (www.ncbi.nlm.nih.gov/blast/).

TABLE 6 DKK oligonucleotide primers for RT-PCR Nucleotide PrimerOrientation Nucleotide sequence (5′ to 3′) position mDkk1F Senseacacacacacacacacacacacatc (SEQ ID NO: 36) 1501-1525 mDkk1R Anti-sensegcaaaagcaccaacccacacttg (SEQ ID NO: 29) 1797-1776 mDkk2F Senseaatgcggaagaatgagggatg (SEQ ID NO: 30) 1593-1613 mDkk2R Anti-sensetgccaatctgaaggaaatgcc (SEQ ID NO: 31) 1839-1819 mDkk3F Sensecgtggacttggcaaaatgtaacc (SEQ ID NO: 32) 1511-1533 mDkk3R Anti-sensegagcactggctttcagaggtattg (SEQ ID NO: 33) 1937-1914 mDkk4F Senseaagccccagaaatcttccagc (SEQ ID NO: 34) 697-717 mDkk4R Anti-sensetgaacacaacaacaagtcccgtg (SEQ ID NO: 35) 839-817 hDkk1F Senseccaacgcgatcaagaacctgcc (SEQ ID NO: 37) 154-176 hDkk1R Anti-sensegatggtgatctttctgtatcc (SEQ ID NO: 38) 790-811 hDkk2F Sensectgatggtggagagctcacag (SEQ ID NO: 39) 201-223 hDkk2R Anti-sensecctgatggagcactggtttgcag (SEQ ID NO: 40) 749-772 hDkk3F Senseagtacacctgccagccatg (SEQ ID NO: 41) 611-630 hDkk3R Anti-sensectccaggtcttccagctcctgg (SEQ ID NO: 42) 1072-1093 hDkk4F Senseggtcctggacttcaacaacatc (SEQ ID NO: 43) 168-190 hDkk4R Anti-sensecttaatcgagcatgctgccg (SEQ ID NO: 44) 738-758Real-Time Quantitative PCR:

One microgram of total RNA was reverse transcribed into total cDNA.Quantitative PCR (qPCR) was performed using an ABI Prism 7000 sequencedetection system (Applied Biosystems, Foster City, Calif.). The reactionmixture contained 1 ml of cDNA, dedicated buffers with specific primersand probes (5′-labeled by 6-carboxy-fluorescein and 3′-labeled by1-carboxy-teteramethyrhdamine), and DNA polymerase in a total 20 mlvolume. Following 2 min incubation at 50° C. and 10 min incubation at95° C. for denaturing, the reaction was subjected to 40-cycleamplification at 95° C. for 15 s to denature and at 60° C. for 1 min forannealing/extension. Each cDNA sample was analyzed in triplicate inparallel with GAPDH as a control. Changes in mRNA concentration weredetermined by subtracting the CT (threshold cycle) of target gene fromthe CT of GAPDH (Δ=CT gene-CT GAPDH). The mean of Δ control wassubtracted from the ΔSiLRP5/6 reaction (mean Δ control−ΔSiLRP5/6=e) Thedifference was calculated as 2^(e) by the ^(2−ΔΔCT) (Livak andSchmittgen, 2001).

Silencing DKK1 Expression by DKK1 Short Hairpin RNA:

A sequence previously shown be an effective siRNA specific to human DKK1gene (5′-CAATGGTCTGGTACTTATTCCCGAAGGATTAAGTACCAGACCATTGCACC-3′; SEQ IDNO: 45) (Hall et al., 2005) was used to design a syntheticdouble-stranded oligonucleotide sequence for short hairpin RNA (shRNA)knockdown studies, as described (Szule et al., 2006) and designedshDKK1. A control oligonucleotide sequence not matching any sequence inthe human genome(5′-GATCCCCGACACGCGACTTGTACCACTTCAAGAGAGTGGTACAAGTCGGTCGTCTTTTTA-3′; SEQID NO: 46) was used as a control shRNA sequence (designated as shCont).Both double-stranded shRNA sequences were obtained from Integrated DNATechnologies (Coralville, Iowa). The double-stranded oligonucleotideswere cloned into pLVTHM, and virus was generated by cotransfection of293T cells with the pLVTHM vector and helper plasmids pMD2G andpCMV-dR8.91 (all kindly provided by Dr Didier Trono, University ofGeneva, Switzerland). The crude lentivirus was concentrated fromcultured supernatant of the 293T cells and filtered (0.45 μm) and viraltiters were determined by measuring the percent of green fluorescentprotein (GFP)-positive cells present 48 hours after infection of 293Tcells. The Saos-2 and MG63 cells were infected with lentivirussupernatant for indicated times. The efficiency of infection with shDKK1and shCont virus was determined by counting the percent of greenfluorescent protein (GFP) positive cells by fluorescence microscopy.Total RNA, isolated after 24, 48 or 72 hours was subjected to RT-PCR andqPCR to determine of the degree of target gene silencing. After 72 hoursafter infection supernatants of the cells were subject to ELISA analysisto determine DKK1 protein concentration.

Statistical Analysis:

Statistical significance of differences between experimental groups wasanalyzed by a Student's t-test using the Microsoft Excel softwarestatistical package. Significant p values were less than 0.05 bytwo-tailed test.

Wnt3a Induces OPG mRNA and Protein Levels in Osteoblasts:

Wnt3a stimulated OPG mRNA expression in a dose-dependent andtime-dependent fashion in murine mesenchymal osteoblast-precursor C2C12cells (FIG. 43B) as well as in human osteoblast-like Saos-2 cells (FIG.43B) and in MG63 cells (data not shown), reaching increases of 40-fold,2-fold and 1.5-fold, respectively. Similar results were obtained for OPGprotein levels (by ELISA analysis of culture supernatants) (FIG. 43C),with revealed increases relative to controls by 2000-fold in C2C12 cellsversus 4-fold in Saos-2 (FIG. 43D). The response in OPG protein levelwas consistent with free b-catenin levels in the cytoplasm as measuredby E-cadherin binding analysis (FIG. 43C).

DKK1 Diminishes Wnt3a Mediated OPG Production in Osteoblasts:

Using recombinant DKK1 protein, b-catenin level was reduced (using thepull-down assay) in C2C12 (FIG. 43E) and in Saos-2 cells (FIG. 43F).Higher DKK1 concentrations were required for effective DKK1-inducedattenuation of Wnt3a-induced OPG transcription and translation in Saos-2than C2C12 cells (FIGS. 43G and 43H) Although endogenous OPG mRNA andprotein levels were approximately 40-fold and 100-fold higher in Saos-2and MG63 cells, respectively, than in C2C12 cells (see FIGS. 43A to43J), induction of OPG mRNA and protein in response to Wnt3a stimulationin both Saos-2 and MG63 were less obvious than in C2C12 cells,suggesting a greater sensitivity of these cells to DKK1.

Over-Expression of DKK1 in C2C12 Cells Reduces Wnt3-Induced OPG:

The present invention examined differences in response to Wnt3astimulation relative to DKK1 concentrations required for Wnt3ainhibition between these cell lines. Examining endogenous DKK mRNAstatus by RT-PCR analysis across cell lines revealed that C2C12 cellshad lower levels of DKK1 than DKK2 and DKK3 (FIG. 43K), whereas therewas abundance of all DKK (including DKK1) mRNAs in three humanosteoblast lines (Saos-2, MG63, hFOB1.19) (FIG. 43L). Similar resultswere obtained with DKK protein measurements (FIG. 43M), suggesting thatthe relatively high endogenous DKK1 protein levels in Saos-2 and MG63cells may interfere with the ability of these cells to respond to Wnt3asimulation. To test this hypothesis, C2C12 cells were transfected withconstructs containing Dkk1 cDNA (pEF/DKK1) or empty vector (pEF/EV), andDKK1 protein levels were detected in these stable clones by anti-V5antibody (FIG. 43N). Significantly higher concentrations of DKK1 protein(160 ng/ml) in pEF/DKK1 clones were detected by ELISA analysis comparedwith vector control (pEF/EV) cells (FIG. 43O). OPG mRNA (FIG. 43P) andOPG protein (FIG. 43Q) were both significantly reduced inDKK1-expressing C2C12 cells (pEF/DKK1) compared to control cells. Theseresults suggest C2C12 cells, upon DKK1 transfection, become lesssensitive to Wnt3a signaling and thus become more similar to the humanosteoblast-like cells.

DKK1 Silencing by shRNA Restores Sensitivity to Wnt3a Stimulation inSaos-2 Cells:

To further confirm that impaired Wnt3a signaling can be related toendogenous DKK1, DKK1-specific shRNA silencing experiments were carriedout. Endogenous DKK1 mRNA in Saos-2 cells was inhibited shDKK1, asdetermined by RT-PCR, but not by a non-specific shRNA (FIG. 43R).Relative to shCont cells, a time-dependent significant decrease in DKK1protein levels was observed in shDKK1-expressing Saos-2 cells (FIG.43S); such cells responded to Wnt3a treatment with a significantincrease in OPG mRNA (FIG. 43T) and OPG protein (FIG. 43U) relative tocontrols. Thus, endogenous DKK1 levels critically control theresponsiveness of Wnt3a signaling in osteoblasts as measured by OPGproduction. The low endogenous levels of DKK1 in C2C12 cells makes thesecells particularly well suited to investigate the role of Wnt3a exposureon OPG expression in cells of the osteoblast lineage.

Co-Culture with MM Cells Expressing DKK1 Prevents Wnt3a-Induced OPG inOsteoblasts:

To determine whether DKK1 expression by MM cells interferes withWnt3a-induced OPG transcription in the bone marrow microenvironment,OPM-2 mM cells stably expressing DKK1 were produced as confirmed bydemonstrating an inhibition of TCF/LEF transcriptional activity relativeto controls (Qiang et al., 2003). Supernatants of OPM-2/DKK1 clonescontained the DKK1 protein as determined by Western blot analysis withanti-V5 antibody (FIG. 43W). ELISA analysis showed that DKK1 inOPM-2/DKK1 expressing clones was significantly higher than that incontrol cells (FIG. 43X). C2C12 cells, co-cultured with DKK1/OPM-2cells, showed significant inhibition of both Wnt3a-induced OPG mRNAexpression, determined by qPCR analysis (FIG. 43Y) and of OPG protein at48 and 72 hours (FIG. 43Z).

The same experiment was repeated with DKK1-expressing primary MM plasmacells from 5 patients. Results were similar to those obtained withOPM-2/DKK1 cells: a Wnt3a-induced OPG increase was significantlyinhibited in C2C12 cells both at the mRNA (FIG. 43AA) and protein (FIG.43BB) levels in all five cases. Collectively, these results suggest thatDKK1-expressing MM cells impair Wnt3a-induced OPG production inosteoblasts.

Neutralization of DKK1 Protein Restored OPG Levels in Osteoblasts:

Previous studies have shown that DKK1 in sera from MM patients inhibitsosteoblast differentiation (Tian et al., 2003) and bone formation(Giuliani et al., 2007) which was shown to occur through a DKK1-mediatedattenuation of Wnt3a-induced stabilization of b-catenin. Similar to thepresence of 100 ng/ml of DKK1 in culture media, treatment of C2C12 cellswith sera from bone marrow of eight MM patients containing high levelsof DKK1, all in excess of 100 ng/ml of DKK1 (designated MMSH),significantly inhibited Wnt3a-induced increase in OPG mRNA (FIG. 43CC).The observation that sera containing less than 10 ng/ml of DKK1 protein(MMSL) still inhibited Wnt3a-induced OPG transcription might suggestthat factors besides DKK1 may contribute to interference withWnt3a-induced OPG expression.

To verify that DKK1 in sera of MM patients was contributing to thesuppression of Wnt3a-mediated OPG expression in osteoblasts, the MMserum was preincubated with a neutralizing antibody specific to DKK1.Compared to control IgG antibody, pretreatment of C2C12 cells withanti-DKK1 antibody significantly rescued Wnt3a-induced OPG mRNA (FIGS.43DD and 43EE) and protein expression (FIG. 43FF). Collectively, theseresults suggest that DKK1-expressed by MM cells can negatively regulateWnt3a-mediated OPG secretion in osteoblasts.

Wnt3a-Mediated Inhibition of RANKL is Blocked by Dkk1 from MM Cells:

Since indirect activation of Wnt signaling by inhibition of GSK3beta hasbeen reported to regulate RANKL in MC3T3-E1 osteoblasts (Spencer et al.,2006), the effect of DKK1 on this process was examined herein as anotherpotential mechanism underlying MM bone disease. Treatment of C2C12 cellswith Wnt3a for 48 hours resulted in a significant decrease in RANKL mRNA(FIG. 43GG), which could be restored by pretreatment of cells with DKK1.Similar results were observed in DKK1-pretreated Saos-2 (FIG. 43HH) andMG63 cells (FIG. 43II). To further confirm the role of DKK1 on thisprocess, we employed DKK1-overexpressing C2C12 cells, in which high DKK1concentrations can abrogate Wnt3a signaling (see FIG. 43O). RANKL mRNA(FIG. 43JJ) and protein expression (FIG. 43KK) in PEF/DKK1 cells wassignificantly higher than in control pEF/EV in the absence of Wnt3aprotein. Wnt3a treatment of pEF/DKK1 cells significantly altered RANKLexpression (p<0.01), compared with pEF/DKK1 cells without Wnt3a. Theseresults suggest that RANKL protein in osteoblast cells is determined bythe ratio of Wnt and DKK1. We were able to show that sera from 8 mMsubjects with high DKK1 concentrations (>=100 ng/ml) significantlydecreased Wnt3a-mediated inhibition of RANKL mRNA expression (FIG. 43LL)while sera containing low concentration of this molecule (<10 ng/ml)were ineffective. Taken together, these results suggest that DKK1,through inhibition of canonical Wnt signaling, increases RANKL inosteoblasts.

EXAMPLE 20 Effects of Neutralizing Antibody Against DKK1 in thePreclinical SCID-Rab Model for Primary Human Myeloma

Recent clinical and experimental studies suggest that myeloma bonedisease drives tumor progression. Growth of myeloma cells from a subsetof patients was inhibited by inhibitors of osteoclast activity (Yaccobyet al., 2002). Although isolated osteoclasts support survival andproliferation of myeloma cells, osteoblasts have a negative impact onmyeloma. Additionally, studies focusing on cell-signaling molecules havedemonstrated that myeloma cells produce the Wnt signaling inhibitor DKK1that inhibits osteoblast differentiation in vitro (Tian et al., 2001)and that immature as opposed to mature, osteoblasts produce elevatedlevels of RANKL and IL-6 (Gunn et al., 2004). Moreover, synthesis ofosteoprotegerin (OPG), a soluble receptor of RANKL and potent osteoclastinduction signal, is dependent on canonical Wnt signaling in osteoblasts(Glass et al., 2005). Furthermore, DKK1 has been shown to mediatemesenchymal stem cell proliferation in favor of differentiation (Gregoryet al., 2003).

Therefore, whether inhibition of Wnt signaling and osteoblastdifferentiation by DKK1 resulted in increased activity of osteoblastprecursors that induced a cascade of events leading to myeloma diseaseprogression was examined. Additionally, shifts in bone marrowconcentrations of secreted factors DKK1, RANKL, OPG and IL-6 contributesto myeloma cell growth and an absolute shift in numbers of mature andimmature osteoblasts and osteoclasts that favors bone destruction andmyeloma cell growth.

A neutralizing antibody against DKK1 was used in a xenograft SCID-rabmouse model for primary human myeloma (Yata & Yaccoby, 2004) to examinethe effect of DKK1 inhibition on myeloma-induced bone disease and theassociation between increased osteoblast activity and tumor growth. Thissystem is a second generation of the SCID-Hu model (Yaccoby et al.,1998). In these systems, myeloma cells from patients with myelomaengraft in transplanted bone and produce typical disease manifestationsincluding induction of osteolystic bone lesions.

Briefly, SCID-rab host mice were constructed by subcutaneousimplantation of rabbit bones (FIG. 44) as described (Yata and Yaccoby,2004). After 6-8 weeks, myeloma cells from 7 patients were inoculateddirectly into the implanted bone in the host. Tumor growth was thenmonitored by measuring the levels of human monoclonal immunoglobulins inthe mice sera. Increased tumor burden was usually associated withinduction of osteolytic bone lesions as indicated on X-ray radiographs.Treatment was initiated when the levels were higher than 100 μg/ml.However, since the tumor burden varied between patients, treatment ineach experiment was started at different time points after inoculation.

For each patient's cells, one SCID-rab mouse with established myelomawas injected with anti-DKK1 antibodies (R&D Systems) into thesurrounding area of the implanted bone and another served as control andreceived a non-specific IgG antibody. The mice received polyclonalanti-DKK1 antibody at a concentration of 50 μg/injection/3 times a weekin 4 experiments. In 3 experiments, the experimental mice receivedmonoclonal anti-DKK1 antibody at concentration of 100 m/injection/5times a week. Experiments were continued for 4-6 weeks. No drug-relatedtoxicity was observed during the experimental period. The growth ofmyeloma cells, bone resorption and formation, osteoclast and osteoblastnumbers were then determined. The effect of treatment on bone mineraldensity (BMD) and tumor burden were analyzed using Student pairedt-test.

The osteoclast numbers were determined by staining rabbit bone sectionsfor TRAP and TRAP-expressing multinucleated osteoclasts were counted in4 non-overlapping myelomatous bone surface areas of control andanti-DKK1 treated mice. Additionally, mature osteoblasts were identifiedby immunohistochemical staining of rabbit bone sections for osteocalcinand osteoblast numbers were counted in 4 non-overlapping myelomatousbone surface areas of these mice.

Treatment with anti-DKK1 resulted in increased number ofosteocalcin-expressing osteoblasts and reduced TRAP-expressingosteoclasts (FIG. 45). The effect of anti-DKK1 treatment on osteoblastto osteoclast ratios was demonstrated on sequential bone sections. Thesurface of control IgG treated myelomatous bone was characterized byincreased osteoclast activity and a reduction in osteoblasts whereastreatment with anti-DKK1 antibody led to an increased osteoblast numbersand reduced osteoclast numbers on the same bone surface (FIG. 46).

Next, whether anti-DKK1 effect on the osteoclast and osteoblast activityaffected myeloma-induced bone loss in these mice was assessed. Boneresorption and formation was visualized by X-ray radiographs andquantified by measuring bone mineral density (BMD) of the implanted bonebefore the start of the treatment and at the end of each experiment. Incontrol mice, the implanted rabbit bone mineral density was reducedduring the experimental period. The bone mineral density in bonestreated with anti-DKK1 was increased by >8% from pretreatment level(p<0.04) indicative of increased bone formation (FIG. 47). The boneanabolic affect of anti-DKK1 could also be visualized on x-rayradiographs; whereas in control mice bone resorption and lytic bonelesions were increased during the experimental period, the myelomatousbones from mice treated with anti-DKK1 had increased bone mass (FIG.48).

Furthermore, myeloma tumor burden gradually increased in all controlmice with time. In distinct contrast, an inhibition of tumor burden in 4of 7 experiments and retardation of growth in the other 3 experimentswas observed in mice treated with anti-DKK1 antibody. Overall, myelomagrowth in mice treated with control and anti-DKK1 antibodies increasedby 331% ad 162%, respectively (FIG. 47, p<0.02). Additionally, thegrowth of myeloma cells was also monitored by measuring the levels ofhuman monoclonal immunoglobulins (hIg) in the mice sera and confirmed atthe end of the experiments by histological examinations (H&E, cIg).Histological examination revealed that myeloma cells were absent in bonearea containing high numbers of osteoblasts due to anti-DKK1 treatment(FIG. 49).

EXAMPLE 21 The Effects of Neutralizing Antibody Against DKK1 on BoneMineral Density in the Preclinical Nonmyelomatous SCID-Rab Model

The effects of the neutralizing antibody against DKK1 were determined inSCID-rab mouse, constructed by subcutaneous implantation of rabbit bones(FIG. 44) as described (Yata and Yaccoby, 2004). The mice receivedpolyclonal anti-DKK1 antibody at a concentration of 50 μg/injection/3times a week in 4 experiments. In 3 experiments, the experimental micereceived monoclonal anti-DKK1 antibody at concentration of 100μg/injection/5 times a week. Experiments were continued for 4-6 weeks.The effects of DKK1 neutralizing antibody on the bone marrow density ofimplanted femurs in nonmyelomatous mice (n=18), and the uninvolvedmurine femur of myelomatous SCID-Rab mice (n=9) was determined.Treatment with DKK1 antibody resulted in a significant increase in bonemarrow density of the nonmyelomatous implanted bone relative to controls(treated with irrelevant IgG antibody for 4-6 weeks) (19±6% vs. 3±5%;p<0.05) and in the murine femur (4.4±0.6% vs. 0.1±1.4%; p<0.015) (FIGS.50A-50C). The bone marrow density of uninvolved mouse femurs frommyelomatous hosts did not change in both DKK1 AB-treated and controls(4.0±3.2% vs. 3.4±2.5%) (FIGS. 50A-50C).

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method of increasing osteoblast activity in a subject exhibiting increased DKK1 activity or expression, comprising the step of: administering to the subject an agent that is a DKK1 neutralizing antibody that binds a DKK1 protein consisting of the sequence encoded by the DKK1 gene consisting of the sequence shown in SEQ ID No: 47, wherein the agent inhibits the activity of the DKK1 protein.
 2. A method of inhibiting osteoclast activity in a subject, exhibiting increased DKK1 activity or expression, comprising the step of: administering to the subject an agent that is a DKK1 neutralizing antibody that binds a DKK1 protein consisting of the sequence encoded by the DKK1 gene consisting of the sequence shown in SEQ ID No: 47, wherein the agent inhibits the activity of the DKK1 protein.
 3. A method of increasing bone mass in a subject, exhibiting increased DKK1 activity or expression, comprising the step of: administering to the subject an agent that is a DKK1 neutralizing antibody that binds a DKK1 protein consisting of the sequence encoded by the DKK1 gene consisting of the sequence shown in SEQ ID No: 47, wherein the agent inhibits the activity of the DKK1 protein. 